Subfamily e simian adenoviruses a1321, a1325, a1295, a1309, a1316 and a1322 and uses thereof

ABSTRACT

Recombinant vectors comprise simian adenovirus A1321 (SAdV-A1321), SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, and/or SAdV-A1322 sequences and a heterologous gene under the control of regulatory sequences. A cell line which expresses simian adenovirus SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, and/or SAdV-A1322 gene(s) is also disclosed. Methods of using the vectors and cell lines are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/475,535, filed May 18, 2012, which is a continuation-in-part ofPCT/US2011/061632, filed Nov. 21, 2011, which claims the benefit under35 USC 119(e) of U.S. Patent Application Nos. 61/416,467, filed Nov. 23,2010 (now expired), 61/416,481, filed Nov. 23, 2010 (now expired),61/416,491, filed Nov. 23, 2010 (now expired), 61/416,499, filed Nov.23, 2010 (now expired), 61/416,509, filed Nov. 23, 2010 (now expired),and 61/416,515, filed Nov. 23, 2010 (now expired), all of which areincorporated by reference herein.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeled“X5708D1USA_ST25.txt”.

BACKGROUND OF THE INVENTION

Adenovirus is a double-stranded DNA virus with a genome size of about 36kilobases (kb), which has been widely used for gene transferapplications due to its ability to achieve highly efficient genetransfer in a variety of target tissues and large transgene capacity.Conventionally, E1 genes of adenovirus are deleted and replaced with atransgene cassette consisting of the promoter of choice, cDNA sequenceof the gene of interest and a poly A signal, resulting in a replicationdefective recombinant virus.

Adenoviruses have a characteristic morphology with an icosahedral capsidconsisting of three major proteins, hexon (II), penton base (III) and aknobbed fibre (IV), along with a number of other minor proteins, VI,VIII, IX, IIIa and IVa2 [W. C. Russell, J. Gen Virol., 81:2573-3704(November 2000)]. The virus genome is a linear, double-stranded DNA witha terminal protein attached covalently to the 5′ terminus, which haveinverted terminal repeats (ITRs). The virus DNA is intimately associatedwith the highly basic protein VII and a small peptide pX (formerlytermed mu). Another protein, V, is packaged with this DNA-proteincomplex and provides a structural link to the capsid via protein VI. Thevirus also contains a virus-encoded protease, which is necessary forprocessing of some of the structural proteins to produce matureinfectious virus.

A classification scheme has been developed for the Mastadenovirusfamily, which includes human, simian, bovine, equine, porcine, ovine,canine and opossum adenoviruses. This classification scheme wasdeveloped based on the differing abilities of the adenovirus sequencesin the family to agglutinate red blood cells. The result was sixsubgroups, now referred to as subgroups A, B, C, D, E and F. See, T.Shenk et al., Adenoviridae: The Viruses and their Replication”, Ch. 67,in FIELD'S VIROLOGY, 6^(th) Ed., edited by B. N Fields et al,(Lippincott Raven Publishers, Philadelphia, 1996), p. 111-2112.

Recombinant adenoviruses have been described for delivery ofheterologous molecules to host cells. See, U.S. Pat. No. 6,083,716,which describes the genome of two chimpanzee adenoviruses. Simianadenoviruses, C5, C6 and C7, have been described in U.S. Pat. No.7,247,472 as being useful as vaccine vectors. Other chimpanzeeadenoviruses are described in WO 2005/1071093 as being useful for makingadenovirus vaccine carriers.

What is needed in the art are vectors which effectively delivermolecules to a target and minimize the effect of pre-existing immunityto selected adenovirus serotypes in the population.

SUMMARY OF THE INVENTION

Isolated nucleic acid sequences and amino acid sequences of six novelsubfamily E simian adenoviruses, and vectors containing these sequences,are provided herein. Also provided are a number of methods for using thevectors and cells of the invention. These adenoviruses includeSAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, andSAdV-A1322.

The methods described herein involve delivering one or more selectedheterologous gene(s) to a mammalian patient by administering a vector ofthe invention. Use of the compositions described herein for vaccinationpermits presentation of a selected antigen for the elicitation ofprotective immune responses. The vectors based on these simianadenoviruses may also be used for producing heterologous gene productsin vitro. Such gene products are themselves useful for a variety ofpurposes such as are described herein.

These and other embodiments and advantages of the invention aredescribed in more detail below.

DETAILED DESCRIPTION OF THE INVENTION

Novel nucleic acid and amino acid sequences from simian adenovirusA1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, and SAdV-A1322,all of which were isolated from chimpanzee feces, are provided.

Also provided are novel adenovirus vectors and packaging cell lines toproduce vectors based on these sequences for use in the in vitroproduction of recombinant proteins or fragments or other reagents.Further provided are compositions for use in delivering a heterologousmolecule for therapeutic or vaccine purposes. Such therapeutic orvaccine compositions contain the adenoviral vectors carrying an insertedheterologous molecule. In addition, the novel SAdV sequences are usefulin providing the essential helper functions required for production ofrecombinant adeno-associated viral (AAV) vectors. Thus, helperconstructs, methods and cell lines which use these sequences in suchproduction methods, are provided.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95 to 99%, includingabout 96%, about 97%, about 98%, and about 99% of the aligned sequences.

The term “substantial homology” or “substantial similarity,” whenreferring to amino acids or fragments thereof, indicates that, whenoptimally aligned with appropriate amino acid insertions or deletionswith another amino acid (or its complementary strand), there is aminoacid sequence identity in at least about 95 to 99% %, including about96%, about 97%, about 98%, and about 99%, of the aligned sequences.Preferably, the homology is over full-length sequence, or a proteinthereof, or a fragment thereof which is at least 8 amino acids, or moredesirably, at least 15 amino acids in length. Examples of suitablefragments are described herein.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences thatare the same when aligned for maximum correspondence. Where gaps arerequired to align one sequence with another, the degree of scoring iscalculated with respect to the longer sequence without penalty for gaps.Sequences that preserve the functionality of the polynucleotide or apolypeptide encoded thereby are more closely identical. The length ofsequence identity comparison may be over the full-length of the genome(e.g., about 36 kbp), the full-length of an open reading frame of agene, protein, subunit, or enzyme [see, e.g., the tables providing theadenoviral coding regions], or a fragment of at least about 500 to 5000nucleotides, is desired. However, identity among smaller fragments, e.g.of at least about nine nucleotides, usually at least about 20 to 24nucleotides, at least about 28 to 32 nucleotides, at least about 36 ormore nucleotides, may also be desired. Similarly, “percent sequenceidentity” may be readily determined for amino acid sequences, over thefull-length of a protein, or a fragment thereof. Suitably, a fragment isat least about 8 amino acids in length, and may be up to about 700 aminoacids. Examples of suitable fragments are described herein.

Identity is readily determined using such algorithms and computerprograms as are defined herein at default settings. Preferably, suchidentity is over the full length of the protein, enzyme, subunit, orover a fragment of at least about 8 amino acids in length. However,identity may be based upon shorter regions, where suited to the use towhich the identical gene product is being put.

As described herein, alignments are performed using any of a variety ofpublicly or commercially available Multiple Sequence Alignment Programs,such as “Clustal W”, accessible through Web Servers on the internet[Thompson et al, 1994, Nucleic Acids Res, 22, 4673-4680]. Alternatively,Vector NTIO utilities [InVitrogen] are also used. There are also anumber of algorithms known in the art that can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta, a program in GCG Version 6.1. Fasta providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta with its default parameters (a word size of 6 and the NOPAM factorfor the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference. Similarly programs are available forperforming amino acid alignments. Generally, these programs are used atdefault settings, although one of skill in the art can alter thesesettings as needed. Alternatively, one of skill in the art can utilizeanother algorithm or computer program that provides at least the levelof identity or alignment as that provided by the referenced algorithmsand programs.

“Recombinant”, as applied to a polynucleotide, means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

Typically, “heterologous” means derived from a genotypically distinctentity from that of the rest of the entity to which it is beingcompared. A heterologous nucleic acid sequence refers to any nucleicacid sequence that is not isolated from, derived from, or based upon anaturally occurring nucleic acid sequence of the adenoviral vector.“Naturally occurring” means a sequence found in nature and notsynthetically prepared or modified. A sequence is “derived” from asource when it is isolated from a source but modified (e.g., bydeletion, substitution (mutation), insertion, or other modification) soas not to disrupt the normal function of the source gene. A sequence is“based upon” a source when the sequence is substantially similar to thesource.

For example, a polynucleotide introduced by genetic engineeringtechniques into a plasmid or vector derived from a different species(and often a different genus, subfamily or family) is a heterologouspolynucleotide. A promoter removed from its native coding sequence andoperatively linked to a coding sequence with which it is not naturallyfound linked is a heterologous promoter. A specific recombination sitethat has been cloned into a genome of a virus or viral vector, whereinthe genome of the virus does not naturally contain it, is a heterologousrecombination site. A heterologous nucleic acid sequence also includes asequence naturally found in an adenoviral genome, but located at anon-native position within the adenoviral vector. When a polynucleotidewith an encoding sequence for a recombinase is used to genetically altera cell that does not normally express the recombinase, both thepolynucleotide and the recombinase are heterologous to the cell.

A heterologous vaccine refers to the situation where one virus or viralvector is introduced in order to induce immunity against a pathogenicvirus of another species. In this case, the term “heterologous” refersan inoculating antigen and challenge antigen derived from viruses havingdifferent species, genus, subfamily, or family specificity.

As used throughout this specification and the claims, the term“comprise” and its variants including, “comprises”, “comprising”, amongother variants, is inclusive of other components, elements, integers,steps and the like. The term “consists of” or “consisting of” areexclusive of other components, elements, integers, steps and the like.

I. The Simian Adenovirus Sequences

The invention provides nucleic acid sequences and amino acid sequencesof simian adenovirus A1321 (SAdV-A1321), SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, and SAdV-A1322, which are each isolated from theother material with which they are associated in nature.

A. Nucleic Acid Sequences

The SAdV-A1321 nucleic acid sequences provided herein includenucleotides 1 to 36546 of SEQ ID NO:1. The SAdV-A1325 nucleic acidsequences herein include nucleotides 1 to 36542 of SEQ ID NO:28. TheSAdV-A1295 nucleic acid sequences provided herein include nucleotides 1to 36643 of SEQ ID NO:57. The SAdV-A1309 nucleic acid sequences providedherein include nucleotides 1 to 36528 of SEQ ID NO:86. The SAdV-A1316nucleic acid sequences provided herein include nucleotides 1 to 36667 ofSEQ ID NO:114. The SAdV-A1322 nucleic acid sequences provided hereininclude nucleotides 1 to 36770 of SEQ ID NO:139. See, Sequence Listing,which is incorporated by reference herein.

In one embodiment, the nucleic acid sequences of the invention furtherencompass the strands which are complementary to the sequences of SEQ IDNO: 1, 28, 57, 86, 114, or 139, respectively, as well as the RNA andcDNA sequences corresponding to the sequences and their complementarystrands. In another embodiment, the nucleic acid sequences furtherencompass sequences which are greater than 98.5% identical, andpreferably, greater than about 99% identical, to the Sequence Listing.Also included in one embodiment, are natural variants and engineeredmodifications of the sequence provided in SEQ ID NO: 1, 28, 57, 86, 114,or 139 and their complementary strands. Such modifications include, forexample, labels that are known in the art, methylation, and substitutionof one or more of the naturally occurring nucleotides with a degeneratenucleotide.

TABLE 1 NUCLEIC ACID REGIONS SAdV-A1321 SAdV-1325 SAdV-A1295 SAdV-A1309SAdV-A1316 SAdV-A1322 ORF ORF ORF ORF ORF ORF SEQ ID SEQ ID SEQ ID SEQID SEQ ID SEQ ID Regions NO: 1 NO: 28 NO: 57 NO: 86 NO: 114 NO:139 ITR1..129 1..129 1..127 1..129 1..128 E1a 13S (576...1140, (882...1146,(577...1141, (576...1143, (577...1144, (576...1140, 12S 1234...1439)1234...1439) 1235...1440) 1228...1433) 1229...1434) 1234...1439)  9S E1bSmall 1601...2155 1604...2173 1602...2153 1599...2177 1605...2186 T/19KLarge 1906...3396 1909...3414 1907...3394 1904...3418 1905...34161910...3427 T/55K IX 3484...3909 3452...3922 3481...3906 3456...39263504...3929 3512...3940 E2b pTP Complement Complement ComplementComplement Complement Complement (8457...10385, (8453...10378,(8448...10376, (8462...10393, (8480...10408, (8479...10422,13829...13837) 13818...13826) 13809...13817) 13849...13857)13841...13849) 13885...13893) Poly- Complement Complement ComplementComplement Complement Complement merase (5077...8655, (5091...8651,(5074...8646, (5094...8660, (5094...8678, (5102...8680, 13829...13837)13818...13826) 13809...13817) 13849...13857) 13841...13849)13885...13893) IVa2 Complement Complement Complement ComplementComplement Complement (3974...5304, (3988...5318, (3971...5301,(3991...5321, (3994...5324, (3999...5332, 5583..5595) 5597...5609)5580...5592) 5600...5612) 5603...5615) 5611...5623) L1 52/55D10809...11984 10806...11984 10850...12022 10841...12019 10873...12054IIIa 12025...13800 12011...13789 12011...13780 12049...1381512046...13812 12078...13859 L2 Penton 13885...15504 13866...1549113857...15458 13897...15516 13897...15846 13938...15566 VII15511...16089 15498...16079 15466...16044 15523...16104 15490...1606815570...16151 V 16134...17147 16127...17167 16092...17117 16152...1719516113...17144 16193...17230 pX 17174...17404 17193...17423 17141...1737117223...17453 17168...17398 17251...17484 L3 VI 17439...1820017495...18220 17407...18180 17525...18253 17433...18206 17516...18289Hexon 18309...21134 18260...21136 18290...21109 18348...2116418312...21125 18396...21218 Endo- 21161...21781 21161...2178121130...21756 21189...21809 21152...21784 21240...21869 protease E2a DBPComplement Complement Complement Complement Complement Complement(21862...23391) (21867...23399) (21838...23373) (21898...23433)(21864...23399) (21948...23486) L4 100 kD 23420...25828 23425...2581223399...25819 23456...25843 23428...25830 23509...25902 22 kD25545...26105 25532...26092 25533..26096 25560...26129 25625...26167VIII 26453...27133 26430...27110 26453...27133 26469...2714926452...27132 26510...27190 E3 12.5K 27114...27431 27137...2754527153...27470 27194...27511 CR1-alpha 27411...28046 27388...2801427411...28043 27427...28050 27410...28042 27468...28088 gp19K28031...28558 27999...28526 28028...28555 28035...28565 28073...28597CR1-beta 28591...29193 28559...29176 28589...29317 28598...2921528532...29323 28637...29356 CR1-gamma 29209...29823 29192...2980029333...29953 29231...29845 29339...29962 29372...29992 CR1-delta29819...30736 29818...30681 29976...30848 29863...30741 29985...3088129854...30891 RID-alpha 30748...31020 30692...30964 30860...3113230893...31165 30902...31174 RID-beta 31029...31457 30973...3140431135...31572 31033...31464 31174...31602 31183...31614 14.7K31453...31854 31400...31804 31568...31969 31460...31864 31598...3199931610...32014 L5 Fiber 32117...33451 32101...33429 32227...3355232137...33411 32253...33578 32270...33595 E4 Orf 6/7 ComplementComplement Complement Complement Complement Complement (33550...33801,(33543...33794, (33646...33897, (33523...33774, (33671...33922,(33722...33973, 34524...34697) 34526...34696) 34620...34793)34506...34676) 34645...34818) 34699...34884) Orf 6 Complement ComplementComplement Complement Complement Complement (33801...34697)(33794...34696) (33897...34793) (33774...34676) (33922...34818)(33970...34884) Orf 4 Complement Complement Complement ComplementComplement Complement (34790...35131) (34605...34967) (34669...35064)(34585...34947) (34911...35252) (34775...35140) Orf 3 ComplementComplement Complement Complement Complement Complement (34980...35530)(34980...35330) (35076...35426) (34960...35310) (35101...35451)(35149...35502) Orf 2 Complement Complement Complement ComplementComplement Complement (35330...35716) (35330...35716) (35426...35812)(35310...35696) (35451...35837) (35499...35888) Orf 1 ComplementComplement Complement Complement Complement Complement (35769...36140)(35760...36131) (35865...36236) (35739...36110) (35890...36261)(35939...36316) ITR Complement Complement Complement ComplementComplement Complement (36418...36546) (36413...36542) (36517...36643)(36400...36528) (36540...36667) (36653...36674)

In one embodiment, fragments of the sequences of SEQ ID NO: 1, 28, 57,86, 114, or 139 and their complementary strands, cDNA and RNAcomplementary thereto are provided, along with fragments that havesubstantial homology thereto. Suitable fragments are at least 15nucleotides in length, and encompass functional fragments, i.e.,fragments which are of biological interest. For example, a functionalfragment can express a desired adenoviral product or may be useful inproduction of recombinant viral vectors. Such fragments include the genesequences and fragments listed in the tables herein. The tables providethe transcript regions and open reading frames in the SAdV-A1321,SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, and SAdV-A1322sequences. For certain genes, the transcripts and open reading frames(ORFs) are located on the strand complementary to that presented in SEQID NO: 1, 28, 57, 86, 114, or 139. See, e.g., E2a, E2b, and E4. Thecalculated molecular weights of the encoded proteins are also shown.Note that the E1a open reading frame, E2b open reading frame, and E4open reading frame contain internal splice sites. These splice sites arenoted in the table above.

The SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 adenoviral nucleic acid sequences are useful as therapeuticagents and in construction of a variety of vector systems and hostcells. As used herein, a vector includes any suitable nucleic acidmolecule including, naked DNA, a plasmid, a virus, a cosmid, or anepisome. These sequences and products may be used alone or incombination with other adenoviral sequences or fragments, or incombination with elements from other adenoviral or non-adenoviralsequences. The SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 sequences are also useful as antisensedelivery vectors, gene therapy vectors, or vaccine vectors. Thus,further provided are nucleic acid molecules, gene delivery vectors, andhost cells which contain the SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 sequences.

For example, the invention encompasses a non-naturally occurring nucleicacid molecule containing simian Ad ITR sequences of the invention.“Non-naturally occurring” refers to sequences or genetic elements thatcannot be found in nature and have been synthesized, rearranged, ormodified through recombinant, genetic engineering, or other techniques,along with progeny from vectors and host cells containing same. Inanother example, the invention provides a nucleic acid moleculecontaining simian Ad sequences of the invention encoding a desired Adgene product. Still other nucleic acid molecule constructed using thesequences of the invention will be readily apparent to one of skill inthe art, in view of the information provided herein.

In one embodiment, the simian Ad gene regions identified herein may beused in a variety of vectors for delivery of a heterologous molecule toa cell. For example, vectors are generated for expression of anadenoviral capsid protein (or fragment thereof) for purposes ofgenerating a viral vector in a packaging host cell. Such vectors may bedesigned for expression in trans. Alternatively, such vectors aredesigned to provide cells which stably contain sequences which expressdesired adenoviral functions, e.g., one or more of E1a, E1b, theterminal repeat sequences, E2a, E2b, E4, E4ORF6 region.

In addition, the adenoviral gene sequences and fragments thereof areuseful for providing the helper functions necessary for production ofhelper-dependent viruses (e.g., adenoviral vectors deleted of essentialfunctions, or adeno-associated viruses (AAV)). For such productionmethods, the SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316,or SAdV-A1322 sequences can be utilized in such a method in a mannersimilar to those described for the human Ad. However, due to thedifferences in sequences between the SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 sequences and those of human Ad,the use of the SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 sequences greatly minimize or eliminate thepossibility of homologous recombination with helper functions in a hostcell carrying human Ad E1 functions, e.g., 293 cells, which may produceinfectious adenoviral contaminants during rAAV production.

Methods of producing rAAV using adenoviral helper functions have beendescribed at length in the literature with human adenoviral serotypes.See, e.g., U.S. Pat. No. 6,258,595 and the references cited therein.See, also, U.S. Pat. No. 5,871,982; WO 99/14354; WO 99/15685; WO99/47691. These methods may also be used in production of non-humanserotype AAV, including non-human primate AAV serotypes. The SAdV-A1321,SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 sequenceswhich provide the necessary helper functions (e.g., E1a, E1b, E2a, E2b,DNA polymerase and/or E4 ORF6) can be particularly useful in providingthe necessary adenoviral function while minimizing or eliminating thepossibility of recombination with any other adenoviruses present in therAAV-packaging cell which are typically of human origin. Thus, selectedgenes or open reading frames of the SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 sequences may be utilized in theserAAV production methods.

Alternatively, recombinant SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 vectors may be utilized in thesemethods. Such recombinant adenoviral simian vectors may include, e.g., ahybrid chimp Ad/AAV in which chimp Ad sequences flank a rAAV expressioncassette composed of, e.g., AAV 3′ and/or 5′ ITRs and a transgene underthe control of regulatory sequences which control its expression. One ofskill in the art will recognize that still other simian adenoviralvectors and/or SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 gene sequences will be useful for productionof rAAV and other viruses dependent upon adenoviral helper.

In still another embodiment, nucleic acid molecules are designed fordelivery and expression of selected adenoviral gene products in a hostcell to achieve a desired physiologic effect. For example, a nucleicacid molecule containing sequences encoding an SAdV-A1321, SAdV-A1325,SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 E1a protein may bedelivered to a subject for use as a cancer therapeutic. Optionally, sucha molecule is formulated in a lipid-based carrier and preferentiallytargets cancer cells. Such a formulation may be combined with othercancer therapeutics (e.g., cisplatin, taxol, or the like). Still otheruses for the adenoviral sequences provided herein will be readilyapparent to one of skill in the art.

In addition, one of skill in the art will readily understand that theSAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 sequences can be readily adapted for use for a variety ofviral and non-viral vector systems for in vitro, ex vivo or in vivodelivery of therapeutic and immunogenic molecules. For example, theSAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 simian Ad sequences can be utilized in a variety of rAd andnon-rAd vector systems. Such vectors systems may include, e.g.,plasmids, lentiviruses, retroviruses, poxviruses, vaccinia viruses, andadeno-associated viral systems, among others. Selection of these vectorsystems is not a limitation of the present invention.

The invention further provides molecules useful for production of thesimian and simian-derived proteins of the invention. Such moleculeswhich carry polynucleotides including the simian Ad DNA sequences of theinvention can be in the form of naked DNA, a plasmid, a virus or anyother genetic element.

B. SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 Adenoviral Proteins

Gene products of the SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 adenovirus, such as proteins, enzymes, andfragments thereof, which are encoded by the adenoviral nucleic acidsdescribed herein are provided. Further encompassed are SAdV-A1321,SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 proteins,enzymes, and fragments thereof, having the amino acid sequences encodedby these nucleic acid sequences which are generated by other methods.Such proteins include those encoded by the open reading framesidentified in the table above, the proteins identified in the Tablesbelow with reference to SEQ ID NO, which are provided in the SequenceListing, and sequences that have substantial homology thereto. Fragmentsof the proteins and polypeptides identified herein, along with fragmentshaving substantial homology thereto, are also provided.

TABLE 2 PROTEIN SEQUENCES SAdV-A1321 SAdV- A1325 SAdV-A1295 SAdV-A1309SAdV-A1316 SAdV-A1322 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID RegionsNO: NO: NO: NO: NO: NO: E1a 13S 27 56 85 113 138 167 12S  9S E1b Small 2 29 58  87 140 T/19K Large 21 51 80 108 115 161 T/55K IX  3 30 59  88116 141 L1 52/55D 31 60  89 117 142 IIIa  4 32 61  90 118 143 L2 Penton 5 33 62  91 119 144 VII  6 34 63  92 120 145 V  7 35 64  93 121 146 pX 8 36 65  94 122 147 L3 VI  9 37 66  95 123 148 Hexon 10 38 67  96 124149 Endoprotease 11 39 68  97 125 150 L4 100 kD 12 40 69  98 126 151 22kD 22 52 81 109 162 VIII 13 41 70  99 127 152 E3 12.5k 42 71 100 153CR1-alpha 14 53 82 110 128 163 gp19K 23 43 72 101 154 CR1-beta 15 44 73102 129 155 CR1-gamma 16 45 74 103 130 156 CR1-delta 24 46 75 104 131164 RID-alpha 17 47 76 132 157 RID-beta 18 48 77 105 133 158 14.7K 25 5483 111 136 165 L5 Fiber 19 49 78 106 134 159

Thus, in one aspect, unique simian adenoviral proteins which aresubstantially pure, i.e., are free of other viral and proteinaceousproteins are provided. Preferably, these proteins are at least 10%homogeneous, more preferably 60% homogeneous, and most preferably 95%homogeneous.

In one embodiment, unique simian-derived capsid proteins are provided.As used herein, a simian-derived capsid protein includes any adenoviralcapsid protein that contains a SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 capsid protein or a fragmentthereof, as defined above, including, without limitation, chimericcapsid proteins, fusion proteins, artificial capsid proteins, syntheticcapsid proteins, and recombinant capsid proteins, without limitation tomeans of generating these proteins. A capsid as described herein may beentirely of one of SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322, may contain capsid proteins of more than oneof SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322, or may contain a capsid protein of another adenovirus.

Suitably, these simian-derived capsid proteins contain one or moreSAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 regions or fragments thereof (e.g., a hexon, penton, fiber,or fragment thereof) in combination with capsid regions or fragmentsthereof of different adenoviral serotypes, or modified simian capsidproteins or fragments, as described herein. A “modification of a capsidprotein associated with altered tropism” as used herein includes analtered capsid protein, i.e., a penton, hexon or fiber protein region,or fragment thereof, such as the knob domain of the fiber region, or apolynucleotide encoding same, such that specificity is altered. Thesimian-derived capsid may be constructed with one or more of the simianAd of the invention or another Ad serotype which may be of human ornon-human origin. Such Ad may be obtained from a variety of sourcesincluding the ATCC, commercial and academic sources, or the sequences ofthe Ad may be obtained from GenBank or other suitable sources.

The amino acid sequences of the penton proteins of SAdV-A1321 [SEQ IDNO: 5], SAdV-A1325 [SEQ ID NO: 33], SAdV-A1295 [SEQ ID NO: 62],SAdV-A1309 [SEQ ID NO: 91], SAdV-A1316 [SEQ ID NO: 119], or SAdV-A1322[SEQ ID NO: 144], are provided. Suitably, this penton protein, or uniquefragments thereof, may be utilized for a variety of purposes. Examplesof suitable fragments include the penton having N-terminal and/orC-terminal truncations of about 50, 100, 150, or 200 amino acids, basedupon the amino acid numbering provided above and in SEQ ID NO: 5, 33,62, 91, 119, or 144. Other suitable fragments include shorter internal,C-terminal, or N-terminal fragments. Further, the penton protein may bemodified for a variety of purposes known to those of skill in the art.

Also provided is the amino acid sequence of the hexon proteins ofSAdV-A1321 [SEQ ID NO: 10], SAdV-A1325 [SEQ ID NO: 38], SAdV-A1295 [SEQID NO: 67], SAdV-A1309 [SEQ ID NO: 96], SAdV-A1316 [SEQ ID NO: 124], orSAdV-A1322 [SEQ ID NO: 149]. Suitably, this hexon protein, or uniquefragments thereof, may be utilized for a variety of purposes. Examplesof suitable fragments include the hexon having N-terminal and/orC-terminal truncations of about 50, 100, 150, 200, 300, 400, or 500amino acids, based upon the amino acid numbering provided above and inSEQ ID NO: 10, 38, 67, 96, 124, or 149. Other suitable fragments includeshorter internal, C-terminal, or N-terminal fragments. For example, onesuitable fragment the loop region (domain) of the hexon protein,designated DE1 and FG1, or a hypervariable region thereof. Suchfragments include the regions spanning amino acid residues about 125 to443; about 138 to 441, or smaller fragments, such as those spanningabout residue 138 to residue 163; about 170 to about 176; about 195 toabout 203; about 233 to about 246; about 253 to about 374; about 287 toabout 297; and about 404 to about 430 of the simian hexon proteins, withreference to SEQ ID NO: 10, 38, 67, 96, 124, or 149. Other suitablefragments may be readily identified by one of skill in the art. Further,the hexon protein may be modified for a variety of purposes known tothose of skill in the art. Because the hexon protein is the determinantfor serotype of an adenovirus, such artificial hexon proteins wouldresult in adenoviruses having artificial serotypes. Other artificialcapsid proteins can also be constructed using the chimp Ad pentonsequences and/or fiber sequences of the invention and/or fragmentsthereof.

In one embodiment, an adenovirus having an altered hexon proteinutilizing the sequence of the SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 hexon protein may be generated.One suitable method for altering hexon proteins is described in U.S.Pat. No. 5,922,315, which is incorporated by reference. In this method,at least one loop region of the adenovirus hexon is changed with atleast one loop region of another adenovirus serotype. Thus, at least oneloop region of such an altered adenovirus hexon protein is a simian Adhexon loop region of SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322. In one embodiment, a loop region of theSAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 hexon protein is replaced by a loop region from anotheradenovirus serotype. In another embodiment, the loop region of theSAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 hexon is used to replace a loop region from anotheradenovirus serotype. Suitable adenovirus serotypes may be readilyselected from among human and non-human serotypes, as described herein.The selection of a suitable serotype is not a limitation of the presentinvention. Still other uses for the SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 hexon protein sequences will bereadily apparent to those of skill in the art.

The amino acid sequence of the fiber protein of SAdV-A1321 [SEQ ID NO:19], SAdV-A1325 [SEQ ID NO: 49], SAdV-A1295 [SEQ ID NO: 78], SAdV-A1309[SEQ ID NO: 106], SAdV-A1316 [SEQ ID NO: 134], or SAdV-A1322 [SEQ ID NO:159] are provided. Suitably, this fiber protein, or unique fragmentsthereof, may be utilized for a variety of purposes. One suitablefragment is the fiber knob, located within SEQ ID NO: 19, 49, 78, 106,134, or 159. Examples of other suitable fragments include the fiberhaving N-terminal and/or C-terminal truncations of about 50, 100, 150,or 200 amino acids, based upon the amino acid numbering provided in SEQID NO: 19, 49, 78, 106, 134, or 159. Still other suitable fragmentsinclude internal fragments. Further, the fiber protein may be modifiedusing a variety of techniques known to those of skill in the art.

Unique fragments of the proteins of the SAdV-A1321, SAdV-A1325,SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 are at least 8 aminoacids in length. However, fragments of other desired lengths can bereadily utilized. In addition, modifications as may be introduced toenhance yield and/or expression of a SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 gene product, e.g., constructionof a fusion molecule in which all or a fragment of the SAdV-A1321,SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 geneproduct is fused (either directly or via a linker) with a fusion partnerto enhance are provided herein. Other suitable modifications include,without limitation, truncation of a coding region (e.g., a protein orenzyme) to eliminate a pre- or pro-protein ordinarily cleaved and toprovide the mature protein or enzyme and/or mutation of a coding regionto provide a secretable gene product. Still other modifications will bereadily apparent to one of skill in the art. Further encompassed areproteins having at least about 98%, about 99%, about 99.5%, or about99.9 identity to SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 proteins provided herein.

As described herein, vectors of the invention containing the adenoviralcapsid proteins of SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 are particularly well suited for use inapplications in which the neutralizing antibodies diminish theeffectiveness of other Ad serotype based vectors, as well as other viralvectors. The rAd vectors are particularly advantageous inreadministration for repeat gene therapy or for boosting immune response(vaccine titers).

Under certain circumstances, it may be desirable to use one or more ofthe SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 gene products (e.g., a capsid protein or a fragment thereof)to generate an antibody. The term “an antibody,” as used herein, refersto an immunoglobulin molecule which is able to specifically bind to anepitope. The antibodies may exist in a variety of forms including, forexample, high affinity polyclonal antibodies, monoclonal antibodies,synthetic antibodies, chimeric antibodies, recombinant antibodies andhumanized antibodies. Such antibodies originate from immunoglobulinclasses IgG, IgM, IgA, IgD and IgE.

Such antibodies may be generated using any of a number of methods knowin the art. Suitable antibodies may be generated by well-knownconventional techniques, e.g., Kohler and Milstein and the many knownmodifications thereof. Similarly desirable high titer antibodies aregenerated by applying known recombinant techniques to the monoclonal orpolyclonal antibodies developed to these antigens [see, e.g., PCT PatentApplication No. PCT/GB85/00392; British Patent Application PublicationNo. GB2188638A; Amit et al., 1986 Science, 233:747-753; Queen et al.,1989 Proc. Nat'l. Acad. Sci. USA, 86:10029-10033; PCT Patent ApplicationNo. PCT/WO9007861; and Riechmann et al., Nature, 332:323-327 (1988);Huse et al, 1988a Science, 246:1275-1281]. Alternatively, antibodies canbe produced by manipulating the complementarity determining regions ofanimal or human antibodies to the antigen of this invention. See, e.g.,E. Mark and Padlin, “Humanization of Monoclonal Antibodies”, Chapter 4,The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology ofMonoclonal Antibodies, Springer-Verlag (June, 1994); Harlow et al.,1999, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; and Bird et al., 1988, Science 242:423-437.Further provided by the present invention are anti-idiotype antibodies(Ab2) and anti-anti-idiotype antibodies (Ab3). See, e.g., M. Wettendorffet al., “Modulation of anti-tumor immunity by anti-idiotypicantibodies.” In Idiotypic Network and Diseases, ed. by J. Cerny and J.Hiernaux, 1990 J. Am. Soc. Microbiol., Washington D.C.: pp. 203-229].These anti-idiotype and anti-anti-idiotype antibodies are produced usingtechniques well known to those of skill in the art. These antibodies maybe used for a variety of purposes, including diagnostic and clinicalmethods and kits.

Under certain circumstances, it may be desirable to introduce adetectable label or a tag onto a SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 gene product, antibody or otherconstruct of the invention. As used herein, a detectable label is amolecule which is capable, alone or upon interaction with anothermolecule, of providing a detectable signal. Most desirably, the label isdetectable visually, e.g. by fluorescence, for ready use inimmunohistochemical analyses or immunofluorescent microscopy. Forexample, suitable labels include fluorescein isothiocyanate (FITC),phycoerythrin (PE), allophycocyanin (APC), coriphosphine-O (CPO) ortandem dyes, PE-cyanin-5 (PC5), and PE-Texas Red (ECD). All of thesefluorescent dyes are commercially available, and their uses known to theart. Other useful labels include a colloidal gold label. Still otheruseful labels include radioactive compounds or elements. Additionally,labels include a variety of enzyme systems that operate to reveal acolorimetric signal in an assay, e.g., glucose oxidase (which usesglucose as a substrate) releases peroxide as a product which in thepresence of peroxidase and a hydrogen donor such as tetramethylbenzidine (TMB) produces an oxidized TMB that is seen as a blue color.Other examples include horseradish peroxidase (HRP), alkalinephosphatase (AP), and hexokinase in conjunction with glucose-6-phosphatedehydrogenase which reacts with ATP, glucose, and NAD+ to yield, amongother products, NADH that is detected as increased absorbance at 340 nmwavelength.

Other label systems that are utilized in the methods described hereinare detectable by other means, e.g., colored latex microparticles [BangsLaboratories, Indiana] in which a dye is embedded are used in place ofenzymes to form conjugates with the target sequences to provide a visualsignal indicative of the presence of the resulting complex in applicableassays.

Methods for coupling or associating the label with a desired moleculeare similarly conventional and known to those of skill in the art. Knownmethods of label attachment are described [see, for example, Handbook ofFluorescent probes and Research Chemicals, 6th Ed., R. P. M. Haugland,Molecular Probes, Inc., Eugene, Oreg., 1996; Pierce Catalog andHandbook, Life Science and Analytical Research Products, Pierce ChemicalCompany, Rockford, Ill., 1994/1995]. Thus, selection of the label andcoupling methods do not limit this invention.

The sequences, proteins, and fragments of SAdV-A1321, SAdV-A1325,SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 may be produced by anysuitable means, including recombinant production, chemical synthesis, orother synthetic means. Suitable production techniques are well known tothose of skill in the art. See, e.g., Sambrook et al, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor,N.Y.). Alternatively, peptides can also be synthesized by the well knownsolid phase peptide synthesis methods (Merrifield, J. Am. Chem. Soc.,85:2149 (1962); Stewart and Young, Solid Phase Peptide Synthesis(Freeman, San Francisco, 1969) pp. 27-62). These and other suitableproduction methods are within the knowledge of those of skill in the artand are not a limitation of the present invention.

In addition, one of skill in the art will readily understand that theSAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 sequences can be readily adapted for use for a variety ofviral and non-viral vector systems for in vitro, ex vivo or in vivodelivery of therapeutic and immunogenic molecules. For example, in oneembodiment, the simian Ad capsid proteins and other simian adenovirusproteins described herein are used for non-viral, protein-based deliveryof genes, proteins, and other desirable diagnostic, therapeutic andimmunogenic molecules. In one such embodiment, a protein of theinvention is linked, directly or indirectly, to a molecule for targetingto cells with a receptor for adenoviruses. Preferably, a capsid proteinsuch as a hexon, penton, fiber or a fragment thereof having a ligand fora cell surface receptor is selected for such targeting. Suitablemolecules for delivery are selected from among the therapeutic moleculesdescribed herein and their gene products. A variety of linkersincluding, lipids, polyLys, and the like may be utilized as linkers. Forexample, the simian penton protein may be readily utilized for such apurpose by production of a fusion protein using the simian pentonsequences in a manner analogous to that described in Medina-Kauwe L K,et al, Gene Ther. 2001 May; 8(10):795-803 and Medina-Kauwe L K, et al,Gene Ther. 2001 December; 8(23): 1753-1761. Alternatively, the aminoacid sequences of simian Ad protein IX may be utilized for targetingvectors to a cell surface receptor, as described in US Patent Appln20010047081. Suitable ligands include a CD40 antigen, an RGD-containingor polylysine-containing sequence, and the like. Still other simian Adproteins, including, e.g., the hexon protein and/or the fiber protein,may be used for used for these and similar purposes.

Still other SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316,or SAdV-A1322 adenoviral proteins may be used as alone, or incombination with other adenoviral protein, for a variety of purposeswhich will be readily apparent to one of skill in the art. In addition,still other uses for the SAdV adenoviral proteins will be readilyapparent to one of skill in the art.

II. Recombinant Adenoviral Vectors

The compositions described herein include vectors that deliver aheterologous molecule to cells, either for therapeutic or vaccinepurposes. As used herein, a vector may include any genetic elementincluding, without limitation, naked DNA, a phage, transposon, cosmid,episome, plasmid, or a virus. Such vectors contain simian adenovirus DNAof SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 and a minigene. By “minigene” or “expression cassette” ismeant the combination of a selected heterologous gene and the otherregulatory elements necessary to drive translation, transcription and/orexpression of the gene product in a host cell.

Typically, a SAdV-A1321-, SAdV-A1325-, SAdV-A1295-, SAdV-A1309-,SAdV-A1316-, or SAdV-A1322-derived adenoviral vector is designed suchthat the minigene is located in a nucleic acid molecule which containsother adenoviral sequences in the region native to a selected adenoviralgene. The minigene may be inserted into an existing gene region todisrupt the function of that region, if desired. Alternatively, theminigene may be inserted into the site of a partially or fully deletedadenoviral gene. For example, the minigene may be located in the site ofsuch as the site of a functional E1 deletion or functional E3 deletion,among others that may be selected. The term “functionally deleted” or“functional deletion” means that a sufficient amount of the gene regionis removed or otherwise damaged, e.g., by mutation or modification, sothat the gene region is no longer capable of producing functionalproducts of gene expression. If desired, the entire gene region may beremoved. Other suitable sites for gene disruption or deletion arediscussed elsewhere in the application.

For example, for a production vector useful for generation of arecombinant virus, the vector may contain the minigene and either the 5′end of the adenoviral genome or the 3′ end of the adenoviral genome, orboth the 5′ and 3′ ends of the adenoviral genome. The 5′ end of theadenoviral genome contains the 5′ cis-elements necessary for packagingand replication; i.e., the 5′ inverted terminal repeat (ITR) sequences(which function as origins of replication) and the native 5′ packagingenhancer domains (that contain sequences necessary for packaging linearAd genomes and enhancer elements for the E1 promoter). The 3′ end of theadenoviral genome includes the 3′ cis-elements (including the ITRs)necessary for packaging and encapsidation. Suitably, a recombinantadenovirus contains both 5′ and 3′ adenoviral cis-elements and theminigene is located between the 5′ and 3′ adenoviral sequences. ASAdV-A1321-, SAdV-A1325-, SAdV-A1295-, SAdV-A1309-, SAdV-A1316-, orSAdV-A1322-based adenoviral vector may also contain additionaladenoviral sequences.

Suitably, these SAdV-A1321-, SAdV-A1325-, SAdV-A1295-, SAdV-A1309-,SAdV-A1316-, or SAdV-A1322-based adenoviral vectors contain one or moreadenoviral elements derived from the adenoviral genome of the invention.In one embodiment, the vectors contain adenoviral ITRs from SAdV-A1321,SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 andadditional adenoviral sequences from the same adenoviral serotype. Inanother embodiment, the vectors contain adenoviral sequences that arederived from a different adenoviral serotype than that which providesthe ITRs.

As defined herein, a pseudotyped adenovirus refers to an adenovirus inwhich the capsid protein of the adenovirus is from a differentadenovirus than the adenovirus which provides the ITRs.

Further, chimeric or hybrid adenoviruses may be constructed using theadenoviruses described herein using techniques known to those of skillin the art. See, e.g., U.S. Pat. No. 7,291,498.

The selection of the adenoviral source of the ITRs and the source of anyother adenoviral sequences present in vector is not a limitation of thepresent embodiment. A variety of adenovirus strains are available fromthe American Type Culture Collection, Manassas, Va., or available byrequest from a variety of commercial and institutional sources. Further,the sequences of many such strains are available from a variety ofdatabases including, e.g., PubMed and GenBank. Homologous adenovirusvectors prepared from other simian or from human adenoviruses aredescribed in the published literature [see, for example, U.S. Pat. No.5,240,846]. The DNA sequences of a number of adenovirus types areavailable from GenBank, including type Ad5 [GenBank Accession No.M73370]. The adenovirus sequences may be obtained from any knownadenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, andfurther including any of the presently identified human types. Similarlyadenoviruses known to infect non-human animals (e.g., simians) may alsobe employed in the vector constructs of this invention. See, e.g., U.S.Pat. No. 6,083,716.

The viral sequences, helper viruses (if needed), and recombinant viralparticles, and other vector components and sequences employed in theconstruction of the vectors described herein are obtained as describedabove. The DNA sequences of the SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 simian adenovirus of the inventionare employed to construct vectors and cell lines useful in thepreparation of such vectors.

Modifications of the nucleic acid sequences forming the vectors of thisinvention, including sequence deletions, insertions, and other mutationsmay be generated using standard molecular biological techniques and arewithin the scope of this embodiment.

A. The “Minigene”

The methods employed for the selection of the transgene, the cloning andconstruction of the “minigene” and its insertion into the viral vectorare within the skill in the art given the teachings provided herein.

1. The Transgene

The transgene is a nucleic acid sequence, heterologous to the vectorsequences flanking the transgene, which encodes a polypeptide, protein,or other product, of interest. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstransgene transcription, translation, and/or expression in a host cell.

The composition of the transgene sequence will depend upon the use towhich the resulting vector will be put. For example, one type oftransgene sequence includes a reporter sequence, which upon expressionproduces a detectable signal. Such reporter sequences include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, membrane boundproteins including, for example, CD2, CD4, CD8, the influenzahemagglutinin protein, and others well known in the art, to which highaffinity antibodies directed thereto exist or can be produced byconventional means, and fusion proteins comprising a membrane boundprotein appropriately fused to an antigen tag domain from, among others,hemagglutinin or Myc. These coding sequences, when associated withregulatory elements which drive their expression, provide signalsdetectable by conventional means, including enzymatic, radiographic,colorimetric, fluorescence or other spectrographic assays, fluorescentactivating cell sorting assays and immunological assays, includingenzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) andimmunohistochemistry. For example, where the marker sequence is the LacZgene, the presence of the vector carrying the signal is detected byassays for beta-galactosidase activity. Where the transgene is GFP orluciferase, the vector carrying the signal may be measured visually bycolor or light production in a luminometer.

In one embodiment, the transgene is a non-marker sequence encoding aproduct which is useful in biology and medicine, such as proteins,peptides, RNA, enzymes, or catalytic RNAs. Desirable RNA moleculesinclude tRNA, dsRNA, ribosomal RNA, catalytic RNAs, and antisense RNAs.One example of a useful RNA sequence is a sequence which extinguishesexpression of a targeted nucleic acid sequence in the treated animal.

The transgene may be used for treatment, e.g., of genetic deficiencies,as a cancer therapeutic or vaccine, for induction of an immune response,and/or for prophylactic vaccine purposes. As used herein, induction ofan immune response refers to the ability of a molecule (e.g., a geneproduct) to induce a T cell and/or a humoral immune response to themolecule. The invention further includes using multiple transgenes,e.g., to correct or ameliorate a condition caused by a multi-subunitprotein. In certain situations, a different transgene may be used toencode each subunit of a protein, or to encode different peptides orproteins. This is desirable when the size of the DNA encoding theprotein subunit is large, e.g., for an immunoglobulin, theplatelet-derived growth factor, or a dystrophin protein. In order forthe cell to produce the multi-subunit protein, a cell is infected withthe recombinant virus containing each of the different subunits.Alternatively, different subunits of a protein may be encoded by thesame transgene. In this case, a single transgene includes the DNAencoding each of the subunits, with the DNA for each subunit separatedby an internal ribozyme entry site (IRES). This is desirable when thesize of the DNA encoding each of the subunits is small, e.g., the totalsize of the DNA encoding the subunits and the IRES is less than fivekilobases. As an alternative to an IRES, the DNA may be separated bysequences encoding a 2A peptide, which self-cleaves in apost-translational event. See, e.g., M. L. Donnelly, et al, J. Gen.Virol., 78(Pt 1):13-21 (January 1997); Furler, S., et al, Gene Ther.,8(11):864-873 (June 2001); Klump H., et al., Gene Ther., 8(10):811-817(May 2001). This 2A peptide is significantly smaller than an IRES,making it well suited for use when space is a limiting factor. However,the selected transgene may encode any biologically active product orother product, e.g., a product desirable for study.

Suitable transgenes may be readily selected by one of skill in the art.The selection of the transgene is not considered to be a limitation ofthis embodiment.

2. Regulatory Elements

In addition to the major elements identified above for the minigene, thevector also includes conventional control elements necessary which areoperably linked to the transgene in a manner that permits itstranscription, translation and/or expression in a cell transfected withthe plasmid vector or infected with the virus produced by the invention.As used herein, “operably linked” sequences include both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signalsincluding rabbit beta-globin polyA; sequences that stabilize cytoplasmicmRNA; sequences that enhance translation efficiency (e.g., Kozakconsensus sequence); sequences that enhance protein stability; and whendesired, sequences that enhance secretion of the encoded product. Amongother sequences, chimeric introns may be used.

A great number of expression control sequences, including promoterswhich are native, constitutive, inducible and/or tissue-specific, areknown in the art and may be utilized. Examples of constitutive promotersinclude, without limitation, the TBG promoter, the retroviral Roussarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), thecytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see,e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, thedihydrofolate reductase promoter, the β-actin promoter, thephosphoglycerol kinase (PGK) promoter, and the EF1α promoter[Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art. Forexample, inducible promoters include the zinc-inducible sheepmetallothionine (MT) promoter and the dexamethasone (Dex)-induciblemouse mammary tumor virus (MMTV) promoter. Other inducible systemsinclude the T7 polymerase promoter system [WO 98/10088]; the ecdysoneinsect promoter [No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351(1996)], the tetracycline-repressible system [Gossen et al, Proc. Natl.Acad. Sci. USA, 89:5547-5551 (1992)], the tetracycline-inducible system[Gossen et al, Science, 378:1766-1769 (1995), see also Harvey et al,Curr. Opin. Chem. Biol., 2:512-518 (1998)]. Other systems include theFK506 dimer, VP16 or p65 using castradiol, diphenol murislerone, theRU486-inducible system [Wang et al, Nat. Biotech., 15:239-243 (1997) andWang et al, Gene Ther., 4:432-441 (1997)] and the rapamycin-induciblesystem [Magari et al, J. Clin. Invest., 100:2865-2872 (1997)]. Theeffectiveness of some inducible promoters increases over time. In suchcases one can enhance the effectiveness of such systems by insertingmultiple repressors in tandem, e.g., TetR linked to a TetR by an IRES.Alternatively, one can wait at least 3 days before screening for thedesired function. One can enhance expression of desired proteins byknown means to enhance the effectiveness of this system. For example,using the Woodchuck Hepatitis Virus Posttranscriptional RegulatoryElement (WPRE).

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Another embodiment of the transgene includes a transgene operably linkedto a tissue-specific promoter. For instance, if expression in skeletalmuscle is desired, a promoter active in muscle should be used. Theseinclude the promoters from genes encoding skeletal β-actin, myosin lightchain 2A, dystrophin, muscle creatine kinase, as well as syntheticmuscle promoters with activities higher than naturally occurringpromoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Examples ofpromoters that are tissue-specific are known for liver (albumin,Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus corepromoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein(AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), boneosteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bonesialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)),lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998);immunoglobulin heavy chain; T cell receptor chain), neuronal such asneuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol.Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene (Piccioliet al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and theneuron-specific vgf gene (Piccioli et al., Neuron, 15:373-84 (1995)),among others.

Optionally, vectors carrying transgenes encoding therapeutically usefulor immunogenic products may also include selectable markers or reportergenes may include sequences encoding geneticin, hygromicin or purimycinresistance, among others. Such selectable reporters or marker genes(preferably located outside the viral genome to be packaged into a viralparticle) can be used to signal the presence of the plasmids inbacterial cells, such as ampicillin resistance. Other components of thevector may include an origin of replication. Selection of these andother promoters and vector elements are conventional and many suchsequences are available [see, e.g., Sambrook et al, and references citedtherein].

These vectors are generated using the techniques and sequences providedherein, in conjunction with techniques known to those of skill in theart. Such techniques include conventional cloning techniques of cDNAsuch as those described in texts [Sambrook et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.],use of overlapping oligonucleotide sequences of the adenovirus genomes,polymerase chain reaction, and any suitable method which provides thedesired nucleotide sequence.

III. Production of the Viral Vector

In one embodiment, the simian adenoviral plasmids (or other vectors) areused to produce adenoviral vectors. In one embodiment, the adenoviralvectors are adenoviral particles which are replication-defective. In oneembodiment, the adenoviral particles are rendered replication-defectiveby deletions in the E1a and/or E1b genes. Alternatively, theadenoviruses are rendered replication-defective by another means,optionally while retaining the E1a and/or E1b genes. Similarly, in someembodiments, reduction of an immune response to the vector may beaccomplished by deletions in the E2b and/or DNA polymerase genes. Theadenoviral vectors can also contain other mutations to the adenoviralgenome, e.g., temperature-sensitive mutations or deletions in othergenes. In other embodiments, it is desirable to retain an intact E1aand/or E1b region in the adenoviral vectors. Such an intact E1 regionmay be located in its native location in the adenoviral genome or placedin the site of a deletion in the native adenoviral genome (e.g., in theE3 region).

In the construction of useful simian adenovirus vectors for delivery ofa gene to the human (or other mammalian) cell, a range of adenovirusnucleic acid sequences can be employed in the vectors. For example, allor a portion of the adenovirus delayed early gene E3 may be eliminatedfrom the simian adenovirus sequence which forms a part of therecombinant virus. The function of simian E3 is believed to beirrelevant to the function and production of the recombinant virusparticle. Simian adenovirus vectors may also be constructed having adeletion of at least the ORF6 region of the E4 gene, and more desirablybecause of the redundancy in the function of this region, the entire E4region. Still another vector of this invention contains a deletion inthe delayed early gene E2a. Deletions may also be made in any of thelate genes L1 through L5 of the simian adenovirus genome. Similarly,deletions in the intermediate genes IX and IVa₂ may be useful for somepurposes. Other deletions may be made in the other structural ornon-structural adenovirus genes. The above discussed deletions may beused individually, i.e., an adenovirus sequence for use as describedherein may contain deletions in only a single region. Alternatively,deletions of entire genes or portions thereof effective to destroy theirbiological activity may be used in any combination. For example, in oneexemplary vector, the adenovirus sequence may have deletions of the E1genes and the E4 gene, or of the E1, E2a and E3 genes, or of the E1 andE3 genes, or of E1, E2a and E4 genes, with or without deletion of E3,and so on. As discussed above, such deletions may be used in combinationwith other mutations, such as temperature-sensitive mutations, toachieve a desired result.

An adenoviral vector lacking any essential adenoviral sequences (e.g.,E1a, E1b, E2a, E2b, E4 ORF6, L1, L2, L3, L4 and L5) may be cultured inthe presence of the missing adenoviral gene products which are requiredfor viral infectivity and propagation of an adenoviral particle. Thesehelper functions may be provided by culturing the adenoviral vector inthe presence of one or more helper constructs (e.g., a plasmid or virus)or a packaging host cell. See, for example, the techniques described forpreparation of a “minimal” human Ad vector in International PatentApplication WO96/13597, published May 9, 1996, and incorporated hereinby reference.

1. Helper Viruses

Thus, depending upon the simian adenovirus gene content of the viralvectors employed to carry the minigene, a helper adenovirus ornon-replicating virus fragment may be necessary to provide sufficientsimian adenovirus gene sequences necessary to produce an infectiverecombinant viral particle containing the minigene. Useful helperviruses contain selected adenovirus gene sequences not present in theadenovirus vector construct and/or not expressed by the packaging cellline in which the vector is transfected. In one embodiment, the helpervirus is replication-defective and contains a variety of adenovirusgenes in addition to the sequences described above. Such a helper virusis desirably used in combination with an E1-expressing cell line.

Helper viruses may also be formed into poly-cation conjugates asdescribed in Wu et al, J. Biol. Chem., 374:16985-16987 (1989); K. J.Fisher and J. M. Wilson, Biochem. J., 299:49 (Apr. 1, 1994). Helpervirus may optionally contain a second reporter minigene. A number ofsuch reporter genes are known to the art. The presence of a reportergene on the helper virus which is different from the transgene on theadenovirus vector allows both the Ad vector and the helper virus to beindependently monitored. This second reporter is used to enableseparation between the resulting recombinant virus and the helper virusupon purification.

2. Complementation Cell Lines

To generate recombinant simian adenoviruses (Ad) deleted in any of thegenes described above, the function of the deleted gene region, ifessential to the replication and infectivity of the virus, must besupplied to the recombinant virus by a helper virus or cell line, i.e.,a complementation or packaging cell line. In many circumstances, a cellline expressing the human E1 can be used to transcomplement the chimp Advector. This is particularly advantageous because, due to the diversitybetween the chimp Ad sequences of the invention and the human AdE1sequences found in currently available packaging cells, the use of thecurrent human E1-containing cells prevents the generation ofreplication-competent adenoviruses during the replication and productionprocess. However, in certain circumstances, it will be desirable toutilize a cell line which expresses the E1 gene products that can beutilized for production of an E1-deleted simian adenovirus. Such celllines have been described. See, e.g., U.S. Pat. No. 6,083,716.

If desired, one may utilize the sequences provided herein to generate apackaging cell or cell line that expresses, at a minimum, the adenovirusE1 gene from SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316,or SAdV-A1322 under the transcriptional control of a promoter forexpression in a selected parent cell line. Inducible or constitutivepromoters may be employed for this purpose. Examples of such promotersare described in detail elsewhere in this specification. A parent cellis selected for the generation of a novel cell line expressing anydesired SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 gene. Without limitation, such a parent cell line may be HeLa[ATCC Accession No. CCL 2], A549 [ATCC Accession No. CCL 185], HEK 293,KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] and WI-38 [CCL 75]cells, among others. These cell lines are all available from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209. Other suitable parent cell lines may be obtained fromother sources.

Such E1-expressing cell lines are useful in the generation ofrecombinant simian adenovirus E1 deleted vectors. Additionally, oralternatively, cell lines that express one or more simian adenoviralgene products, e.g., E1a, E1b, E2a, and/or E4 ORF6, can be constructedusing essentially the same procedures are used in the generation ofrecombinant simian viral vectors. Such cell lines can be utilized totranscomplement adenovirus vectors deleted in the essential genes thatencode those products, or to provide helper functions necessary forpackaging of a helper-dependent virus (e.g., adeno-associated virus).The preparation of a host cell involves techniques such as assembly ofselected DNA sequences. This assembly may be accomplished utilizingconventional techniques. Such techniques include cDNA and genomiccloning, which are well known and are described in Sambrook et al.,cited above, use of overlapping oligonucleotide sequences of theadenovirus genomes, combined with polymerase chain reaction, syntheticmethods, and any other suitable methods which provide the desirednucleotide sequence.

In still another alternative, the essential adenoviral gene products areprovided in trans by the adenoviral vector and/or helper virus. In suchan instance, a suitable host cell can be selected from any biologicalorganism, including prokaryotic (e.g., bacterial) cells, and eukaryoticcells, including, insect cells, yeast cells and mammalian cells.Particularly desirable host cells are selected from among any mammalianspecies, including, without limitation, cells such as A549, WEHI, 3T3,10T1/2, HEK 293 cells or PERC6 (both of which express functionaladenoviral E1) [Fallaux, F J et al, (1998), Hum Gene Ther, 9:1909-1917],Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyteand myoblast cells derived from mammals including human, monkey, mouse,rat, rabbit, and hamster. The selection of the mammalian speciesproviding the cells is not a limitation of this invention; nor is thetype of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.

3. Assembly of Viral Particle and Transfection of a Cell Line

Generally, when delivering the vector comprising the minigene bytransfection, the vector is delivered in an amount from about 5 μg toabout 100 μg DNA, and preferably about 10 to about 50 μg DNA to about1×10⁴ cells to about 1×10¹³ cells, and preferably about 10⁵ cells.However, the relative amounts of vector DNA to host cells may beadjusted, taking into consideration such factors as the selected vector,the delivery method and the host cells selected.

The vector may be any vector known in the art or disclosed above,including naked DNA, a plasmid, phage, transposon, cosmids, episomes,viruses, etc. Introduction into the host cell of the vector may beachieved by any means known in the art or as disclosed above, includingtransfection, and infection. One or more of the adenoviral genes may bestably integrated into the genome of the host cell, stably expressed asepisomes, or expressed transiently. The gene products may all beexpressed transiently, on an episome or stably integrated, or some ofthe gene products may be expressed stably while others are expressedtransiently. Furthermore, the promoters for each of the adenoviral genesmay be selected independently from a constitutive promoter, an induciblepromoter or a native adenoviral promoter. The promoters may be regulatedby a specific physiological state of the organism or cell (i.e., by thedifferentiation state or in replicating or quiescent cells) or byexogenously-added factors, for example.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation.

Assembly of the selected DNA sequences of the adenovirus (as well as thetransgene and other vector elements into various intermediate plasmids,and the use of the plasmids and vectors to produce a recombinant viralparticle are all achieved using conventional techniques. Such techniquesinclude conventional cloning techniques of cDNA such as those describedin texts [Sambrook et al, cited above], use of overlappingoligonucleotide sequences of the adenovirus genomes, polymerase chainreaction, and any suitable method which provides the desired nucleotidesequence. Standard transfection and co-transfection techniques areemployed, e.g., CaPO₄ precipitation techniques. Other conventionalmethods employed include homologous recombination of the viral genomes,plaquing of viruses in agar overlay, methods of measuring signalgeneration, and the like.

For example, following the construction and assembly of the desiredminigene-containing viral vector, the vector is transfected in vitro inthe presence of a helper virus into the packaging cell line. Homologousrecombination occurs between the helper and the vector sequences, whichpermits the adenovirus-transgene sequences in the vector to bereplicated and packaged into virion capsids, resulting in therecombinant viral vector particles. The current method for producingsuch virus particles is transfection-based. However, the invention isnot limited to such methods.

The resulting recombinant simian adenoviruses are useful in transferringa selected transgene to a selected cell. In in vivo experiments with therecombinant virus grown in the packaging cell lines, the E1-deletedrecombinant simian adenoviral vectors of the invention demonstrateutility in transferring a transgene to a non-simian, preferably a human,cell.

IV. Use of the Recombinant Adenovirus Vectors

The recombinant simian adenovirus A1321 (SAdV-A1321)-, SAdV-A1325-,SAdV-A1295-, SAdV-A1309-, SAdV-A1316-, or SAdV-A1322-based vectors areuseful for gene transfer to a human or non-simian veterinary patient invitro, ex vivo, and in vivo.

The recombinant adenovirus vectors described herein can be used asexpression vectors for the production of the products encoded by theheterologous genes in vitro. For example, the recombinant adenovirusescontaining a gene inserted into the location of an E1 deletion may betransfected into an E1-expressing cell line as described above.Alternatively, replication-competent adenoviruses may be used in anotherselected cell line. The transfected cells are then cultured in theconventional manner, allowing the recombinant adenovirus to express thegene product from the promoter. The gene product may then be recoveredfrom the culture medium by known conventional methods of proteinisolation and recovery from culture.

A SAdV-A1321-, SAdV-A1325-, SAdV-A1295-, SAdV-A1309-, SAdV-A1316-, orSAdV-A1322-derived recombinant simian adenoviral vector provides anefficient gene transfer vehicle that can deliver a selected transgene toa selected host cell in vivo or ex vivo even where the organism hasneutralizing antibodies to one or more AAV serotypes. In one embodiment,the rAAV and the cells are mixed ex vivo; the infected cells arecultured using conventional methodologies; and the transduced cells arere-infused into the patient. These compositions are particularly wellsuited to gene delivery for therapeutic purposes and for immunization,including inducing protective immunity.

More commonly, the SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 recombinant adenoviral vectors will beutilized for delivery of therapeutic or immunogenic molecules, asdescribed below. It will be readily understood for both applications,that the recombinant adenoviral vectors of the invention areparticularly well suited for use in regimens involving repeat deliveryof recombinant adenoviral vectors. Such regimens typically involvedelivery of a series of viral vectors in which the viral capsids arealternated. The viral capsids may be changed for each subsequentadministration, or after a pre-selected number of administrations of aparticular serotype capsid (e.g., one, two, three, four or more). Thus,a regimen may involve delivery of a rAd with a first simian capsid,delivery with a rAd with a second simian capsid, and delivery with athird simian capsid. A variety of other regimens which use the Adcapsids of the invention alone, in combination with one another, or incombination with other adenoviruses (which are preferablyimmunologically non-crossreactive) will be apparent to those of skill inthe art. Optionally, such a regimen may involve administration of rAdwith capsids of other non-human primate adenoviruses, humanadenoviruses, or artificial sequences such as are described herein. Eachphase of the regimen may involve administration of a series ofinjections (or other delivery routes) with a single Ad capsid followedby a series with another capsid from a different Ad source.Alternatively, the SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 vectors may be utilized in regimens involvingother non-adenoviral-mediated delivery systems, including other viralsystems, non-viral delivery systems, protein, peptides, and otherbiologically active molecules.

The following sections will focus on exemplary molecules which may bedelivered via the adenoviral vectors of the invention.

A. Ad-Mediated Delivery of Therapeutic Molecules

In one embodiment, the above-described recombinant vectors areadministered to humans according to published methods for gene therapy.A simian adenoviral vector bearing the selected transgene may beadministered to a patient, preferably suspended in a biologicallycompatible solution or pharmaceutically acceptable delivery vehicle. Asuitable vehicle includes sterile saline. Other aqueous and non-aqueousisotonic sterile injection solutions and aqueous and non-aqueous sterilesuspensions known to be pharmaceutically acceptable carriers and wellknown to those of skill in the art may be employed for this purpose.

The simian adenoviral vectors are administered in sufficient amounts totransduce the target cells and to provide sufficient levels of genetransfer and expression to provide a therapeutic benefit without undueadverse or with medically acceptable physiological effects, which can bedetermined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the retina and other intraoculardelivery methods, direct delivery to the liver, inhalation, intranasal,intravenous, intramuscular, intratracheal, subcutaneous, intradermal,rectal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe transgene or the condition. The route of administration primarilywill depend on the nature of the condition being treated.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectiveadult human or veterinary dosage of the viral vector is generally in therange of from about 100 μL to about 100 mL of a carrier containingconcentrations of from about 1×10⁶ to about 1×10¹⁵ particles, about1×10¹¹ to 1×10¹³ particles, or about 1×10⁹ to 1×10¹² particles virus.Dosages will range depending upon the size of the animal and the routeof administration. For example, a suitable human or veterinary dosage(for about an 80 kg animal) for intramuscular injection is in the rangeof about 1×10⁹ to about 5×10¹² particles per mL, for a single site.Optionally, multiple sites of administration may be delivered. Inanother example, a suitable human or veterinary dosage may be in therange of about 1×10¹¹ to about 1×10¹⁵ particles for an oral formulation.One of skill in the art may adjust these doses, depending on the routeof administration and the therapeutic or vaccinal application for whichthe recombinant vector is employed. The levels of expression of thetransgene, or for an immunogen, the level of circulating antibody, canbe monitored to determine the frequency of dosage administration. Yetother methods for determining the timing of frequency of administrationwill be readily apparent to one of skill in the art.

An optional method step involves the co-administration to the patient,either concurrently with, or before or after administration of the viralvector, of a suitable amount of a short acting immune modulator. Theselected immune modulator is defined herein as an agent capable ofinhibiting the formation of neutralizing antibodies directed against therecombinant vector of this invention or capable of inhibiting cytolyticT lymphocyte (CTL) elimination of the vector. The immune modulator mayinterfere with the interactions between the T helper subsets (T_(H1) orT_(H2)) and B cells to inhibit neutralizing antibody formation.Alternatively, the immune modulator may inhibit the interaction betweenT_(in) cells and CTLs to reduce the occurrence of CTL elimination of thevector. A variety of useful immune modulators and dosages for use ofsame are disclosed, for example, in Yang et al., J. Virol., 70(9)(September, 1996); International Patent Application No. WO96/12406,published May 2, 1996; and International Patent Application No.PCT/US96/03035, all incorporated herein by reference.

1. Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormonesand growth and differentiation factors including, without limitation,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), transforming growth factor (TGF), platelet-derived growth factor(PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one ofthe transforming growth factor superfamily, including TGF, activins,inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1-15, anyone of the heregluin/neuregulin/ARIA/neu differentiation factor (NDF)family of growth factors, nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliaryneurotrophic factor (CNTF), glial cell line derived neurotrophic factor(GDNF), neurturin, agrin, any one of the family ofsemaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor(HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including, e.g., IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors and, interferons,and, stem cell factor, flk-2/flt3 ligand. Gene products produced by theimmune system are also useful in the invention. These include, withoutlimitation, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimericimmunoglobulins, humanized antibodies, single chain antibodies, T cellreceptors, chimeric T cell receptors, single chain T cell receptors,class I and class II MHC molecules, as well as engineeredimmunoglobulins and MHC molecules. Useful gene products also includecomplement regulatory proteins such as complement regulatory proteins,membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1,CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation, including the low density lipoprotein (LDL)receptor, high density lipoprotein (HDL) receptor, the very low densitylipoprotein (VLDL) receptor, and the scavenger receptor. The inventionalso encompasses gene products such as members of the steroid hormonereceptor superfamily including glucocorticoid receptors and estrogenreceptors, Vitamin D receptors and other nuclear receptors. In addition,useful gene products include transcription factors such as jun, fos,max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD andmyogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3,ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins,interferon regulation factor (IRF-1), Wilms tumor protein, ETS-bindingprotein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkheadfamily of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,factor VIII, factor IX, cystathione beta-synthase, branched chainketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionylCoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin cDNA sequence.

Other useful gene products include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides having a non-naturally occurringamino acid sequence containing insertions, deletions or amino acidsubstitutions. For example, single-chain engineered immunoglobulinscould be useful in certain immunocompromised patients. Other types ofnon-naturally occurring gene sequences include antisense molecules andcatalytic nucleic acids, such as ribozymes, which could be used toreduce overexpression of a target.

Reduction and/or modulation of expression of a gene are particularlydesirable for treatment of hyperproliferative conditions characterizedby hyperproliferating cells, as are cancers and psoriasis. Targetpolypeptides include those polypeptides which are produced exclusivelyor at higher levels in hyperproliferative cells as compared to normalcells. Target antigens include polypeptides encoded by oncogenes such asmyb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target polypeptides for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune disease.Other tumor-associated polypeptides can be used as target polypeptidessuch as polypeptides which are found at higher levels in tumor cellsincluding the polypeptide recognized by monoclonal antibody 17-1A andfolate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce self-directed antibodies. T cellmediated autoimmune diseases include Rheumatoid arthritis (RA), multiplesclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin dependentdiabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis,ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease andulcerative colitis. Each of these diseases is characterized by T cellreceptors (TCRs) that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases.

The simian adenoviral vectors of the invention are particularly wellsuited for therapeutic regimens in which multiple adenoviral-mediateddeliveries of transgenes is desired, e.g., in regimens involvingredelivery of the same transgene or in combination regimens involvingdelivery of other transgenes. Such regimens may involve administrationof a SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 simian adenoviral vector, followed by re-administration witha vector from the same serotype adenovirus. Particularly desirableregimens involve administration of a SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 simian adenoviral vector, in whichthe source of the adenoviral capsid sequences of the vector delivered inthe first administration differs from the source of adenoviral capsidsequences of the viral vector utilized in one or more of the subsequentadministrations. For example, a therapeutic regimen involvesadministration of a SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 vector and repeat administration with one ormore adenoviral vectors of the same or different serotypes. In anotherexample, a therapeutic regimen involves administration of an adenoviralvector followed by repeat administration with a SAdV-A1321, SAdV-A1325,SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 vector which has acapsid which differs from the source of the capsid in the firstdelivered adenoviral vector, and optionally further administration withanother vector which is the same or, preferably, differs from the sourceof the adenoviral capsid of the vector in the prior administrationsteps. These regimens are not limited to delivery of adenoviral vectorsconstructed using the SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 simian sequences. Rather, these regimens canreadily utilize other adenoviral sequences, including, withoutlimitation, other simian adenoviral sequences, (e.g., Pan9 or C68, C1,etc), other non-human primate adenoviral sequences, or human adenoviralsequences, in combination with the SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 vectors. Examples of such simian,other non-human primate and human adenoviral serotypes are discussedelsewhere in this document. Further, these therapeutic regimens mayinvolve either simultaneous or sequential delivery of SAdV-A1321,SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 adenoviralvectors in combination with non-adenoviral vectors, non-viral vectors,and/or a variety of other therapeutically useful compounds or molecules.The invention is not limited to these therapeutic regimens, a variety ofwhich will be readily apparent to one of skill in the art.

B. Ad-Mediated Delivery of Immunogenic Transgenes

The recombinant SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 vectors may also be employed as immunogeniccompositions. As used herein, an immunogenic composition is acomposition to which a humoral (e.g., antibody) or cellular (e.g., acytotoxic T cell) response is mounted to a transgene product deliveredby the immunogenic composition following delivery to a mammal, andpreferably a primate. A recombinant simian Ad can contain in any of itsadenovirus sequence deletions a gene encoding a desired immunogen. Thesimian adenovirus is likely to be better suited for use as a liverecombinant virus vaccine in different animal species compared to anadenovirus of human origin, but is not limited to such a use. Therecombinant adenoviruses can be used as prophylactic or therapeuticvaccines against any pathogen for which the antigen(s) crucial forinduction of an immune response and able to limit the spread of thepathogen has been identified and for which the cDNA is available.

Such vaccinal (or other immunogenic) compositions are formulated in asuitable delivery vehicle, as described above. Generally, doses for theimmunogenic compositions are in the range defined above for therapeuticcompositions. The levels of immunity of the selected gene can bemonitored to determine the need, if any, for boosters. Following anassessment of antibody titers in the serum, optional boosterimmunizations may be desired.

Optionally, a vaccinal composition of the invention may be formulated tocontain other components, including, e.g., adjuvants, stabilizers, pHadjusters, preservatives and the like. Such components are well known tothose of skill in the vaccine art. Examples of suitable adjuvantsinclude, without limitation, liposomes, alum, monophosphoryl lipid A,and any biologically active factor, such as cytokine, an interleukin, achemokine, a ligands, and optimally combinations thereof. Certain ofthese biologically active factors can be expressed in vivo, e.g., via aplasmid or viral vector. For example, such an adjuvant can beadministered with a priming DNA vaccine encoding an antigen to enhancethe antigen-specific immune response compared with the immune responsegenerated upon priming with a DNA vaccine encoding the antigen only.

The recombinant adenoviruses are administered in a “an immunogenicamount”, that is, an amount of recombinant adenovirus that is effectivein a route of administration to transfect the desired cells and providesufficient levels of expression of the selected gene to induce an immuneresponse. Where protective immunity is provided, the recombinantadenoviruses are considered to be vaccine compositions useful inpreventing infection and/or recurrent disease.

Alternatively, or in addition, the vectors of the invention may containa transgene encoding a peptide, polypeptide or protein which induces animmune response to a selected immunogen. The recombinant SAdV vectorsdescribed herein are expected to be highly efficacious at inducingcytolytic T cells and antibodies to the inserted heterologous antigenicprotein expressed by the vector.

For example, immunogens may be selected from a variety of viralfamilies. Example of viral families against which an immune responsewould be desirable include, the picornavirus family, which includes thegenera rhinoviruses, which are responsible for about 50% of cases of thecommon cold; the genera enteroviruses, which include polioviruses,coxsackieviruses, echoviruses, and human enteroviruses such as hepatitisA virus; and the genera apthoviruses, which are responsible for foot andmouth diseases, primarily in non-human animals. Within the picornavirusfamily of viruses, target antigens include the VP1, VP2, VP3, VP4, andVPG. Another viral family includes the calcivirus family, whichencompasses the Norwalk group of viruses, which are an importantcausative agent of epidemic gastroenteritis. Still another viral familydesirable for use in targeting antigens for inducing immune responses inhumans and non-human animals is the togavirus family, which includes thegenera alphavirus, which include Sindbis viruses, RossRiver virus, andVenezuelan, Eastern & Western Equine encephalitis, and rubivirus,including Rubella virus. The flaviviridae family includes dengue, yellowfever, Japanese encephalitis, St. Louis encephalitis and tick borneencephalitis viruses. Other target antigens may be generated from theHepatitis C or the coronavirus family, which includes a number ofnon-human viruses such as infectious bronchitis virus (poultry), porcinetransmissible gastroenteric virus (pig), porcine hemagglutinatingencephalomyelitis virus (pig), feline infectious peritonitis virus(cats), feline enteric coronavirus (cat), canine coronavirus (dog), andhuman respiratory coronaviruses, which may cause the common cold and/ornon-A, B or C hepatitis. Within the coronavirus family, target antigensinclude the E1 (also called M or matrix protein), E2 (also called S orSpike protein), E3 (also called HE or hemagglutin-elterose) glycoprotein(not present in all coronaviruses), or N (nucleocapsid). Still otherantigens may be targeted against the rhabdovirus family, which includesthe genera vesiculovirus (e.g., Vesicular Stomatitis Virus), and thegeneral lyssavirus (e.g., rabies).

Within the rhabdovirus family, suitable antigens may be derived from theG protein or the N protein. The family filoviridae, which includeshemorrhagic fever viruses such as Marburg and Ebola virus, may be asuitable source of antigens. The paramyxovirus family includesparainfluenza Virus Type 1, parainfluenza Virus Type 3, bovineparainfluenza Virus Type 3, rubulavirus (mumps virus), parainfluenzaVirus Type 2, parainfluenza virus Type 4, Newcastle disease virus(chickens), rinderpest, morbillivirus, which includes measles and caninedistemper, and pneumovirus, which includes respiratory syncytial virus.The influenza virus is classified within the family orthomyxovirus andis a suitable source of antigen (e.g., the HA protein, the N1 protein).The bunyavirus family includes the genera bunyavirus (Californiaencephalitis, La Crosse), phlebovirus (Rift Valley Fever), hantavirus(puremala is a hemahagin fever virus), nairovirus (Nairobi sheepdisease) and various unassigned bungaviruses. The arenavirus familyprovides a source of antigens against LCM and Lassa fever virus. Thereovirus family includes the genera reovirus, rotavirus (which causesacute gastroenteritis in children), orbiviruses, and cultivirus(Colorado Tick fever, Lebombo (humans), equine encephalosis, bluetongue).

The retrovirus family includes the sub-family oncorivirinal whichencompasses such human and veterinary diseases as feline leukemia virus,HTLVI and HTLVII, lentivirinal (which includes human immunodeficiencyvirus (HIV), simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), equine infectious anemia virus, andspumavirinal). Among the lentiviruses, many suitable antigens have beendescribed and can readily be selected. Examples of suitable HIV and SIVantigens include, without limitation the gag, pol, Vif, Vpx, VPR, Env,Tat, Nef, and Rev proteins, as well as various fragments thereof. Forexample, suitable fragments of the Env protein may include any of itssubunits such as the gp120, gp160, gp41, or smaller fragments thereof,e.g., of at least about 8 amino acids in length. Similarly, fragments ofthe tat protein may be selected. [See, U.S. Pat. No. 5,891,994 and U.S.Pat. No. 6,193,981.] See, also, the HIV and SIV proteins described in D.H. Barouch et al, J. Virol., 75(5):2462-2467 (March 2001), and R. R.Amara, et al, Science, 292:69-74 (6 Apr. 2001). In another example, theHIV and/or SIV immunogenic proteins or peptides may be used to formfusion proteins or other immunogenic molecules. See, e.g., the HIV-1 Tatand/or Nef fusion proteins and immunization regimens described in WO01/54719, published Aug. 2, 2001, and WO 99/16884, published Apr. 8,1999. The invention is not limited to the HIV and/or SIV immunogenicproteins or peptides described herein. In addition, a variety ofmodifications to these proteins has been described or could readily bemade by one of skill in the art. See, e.g., the modified gag proteinthat is described in U.S. Pat. No. 5,972,596. Further, any desired HIVand/or SIV immunogens may be delivered alone or in combination. Suchcombinations may include expression from a single vector or frommultiple vectors. Optionally, another combination may involve deliveryof one or more expressed immunogens with delivery of one or more of theimmunogens in protein form. Such combinations are discussed in moredetail below.

The papovavirus family includes the sub-family polyomaviruses (BKU andJCU viruses) and the sub-family papillomavirus (associated with cancersor malignant progression of papilloma). The adenovirus family includesviruses (EX, AD7, ARD, O.B.) which cause respiratory disease and/orenteritis. The parvovirus family feline parvovirus (feline enteritis),feline panleucopeniavirus, canine parvovirus, and porcine parvovirus.The herpesvirus family includes the sub-family alphaherpesvirinae, whichencompasses the genera simplexvirus (HSVI, HSVII), varicellovirus(pseudorabies, varicella zoster) and the sub-family betaherpesvirinae,which includes the genera cytomegalovirus (HCMV, muromegalovirus) andthe sub-family gammaherpesvirinae, which includes the generalymphocryptovirus, EBV (Burkitts lymphoma), infectious rhinotracheitis,Marek's disease virus, and rhadinovirus. The poxvirus family includesthe sub-family chordopoxvirinae, which encompasses the generaorthopoxvirus (Variola (Smallpox) and Vaccinia (Cowpox)), parapoxvirus,avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, and thesub-family entomopoxvirinae. The hepadnavirus family includes theHepatitis B virus. One unclassified virus which may be suitable sourceof antigens is the Hepatitis delta virus. Still other viral sources mayinclude avian infectious bursal disease virus and porcine respiratoryand reproductive syndrome virus. The alphavirus family includes equinearteritis virus and various Encephalitis viruses.

Immunogens which are useful to immunize a human or non-human animalagainst other pathogens include, e.g., bacteria, fungi, parasiticmicroorganisms or multicellular parasites which infect human andnon-human vertebrates, or from a cancer cell or tumor cell. Examples ofbacterial pathogens include pathogenic gram-positive cocci includepneumococci; staphylococci; and streptococci. Pathogenic gram-negativecocci include meningococcus; gonococcus. Pathogenic entericgram-negative bacilli include enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigella;haemophilus; moraxella; H. ducreyi (which causes chancroid); brucella;Franisella tularensis (which causes tularemia); yersinia (pasteurella);streptobacillus moniliformis and spirillum; Gram-positive bacilliinclude listeria monocytogenes; erysipelothrix rhusiopathiae;Corynebacterium diphtheria (diphtheria); cholera; B. anthracis(anthrax); donovanosis (granuloma inguinale); and bartonellosis.Diseases caused by pathogenic anaerobic bacteria include tetanus;botulism; other clostridia; tuberculosis; leprosy; and othermycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include Typhus fever, Rocky Mountain spottedfever, Q fever, and Rickettsialpox. Examples of mycoplasma andchlamydial infections include: mycoplasma pneumoniae; lymphogranulomavenereum; psittacosis; and perinatal chlamydial infections. Pathogeniceukaryotes encompass pathogenic protozoa and helminthes and infectionsproduced thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans;Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis;schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm)infections.

Many of these organisms and/or toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracis (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communis and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

Administration of the SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 vectors to deliver immunogens against thevariable region of the T cells are anticipated to elicit an immuneresponse including CTLs to eliminate those T cells. In RA, severalspecific variable regions of TCRs which are involved in the disease havebeen characterized. These TCRs include V-3, V-14, V-17 and Vα-17. Thus,delivery of a nucleic acid sequence that encodes at least one of thesepolypeptides will elicit an immune response that will target T cellsinvolved in RA. In MS, several specific variable regions of TCRs whichare involved in the disease have been characterized. These TCRs includeV-7 and Vα-10. Thus, delivery of a nucleic acid sequence that encodes atleast one of these polypeptides will elicit an immune response that willtarget T cells involved in MS. In scleroderma, several specific variableregions of TCRs which are involved in the disease have beencharacterized. These TCRs include V-6, V-8, V-14 and Vα-16, Vα-3C, Vα-7,Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12. Thus, delivery of a recombinantsimian adenovirus that encodes at least one of these polypeptides willelicit an immune response that will target T cells involved inscleroderma.

C. Ad-Mediated Delivery Methods

The therapeutic levels, or levels of immunity, of the selected gene canbe monitored to determine the need, if any, for boosters. Following anassessment of CD8+T cell response, or optionally, antibody titers, inthe serum, optional booster immunizations may be desired. Optionally,the recombinant SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 vectors may be delivered in a singleadministration or in various combination regimens, e.g., in combinationwith a regimen or course of treatment involving other active ingredientsor in a prime-boost regimen. A variety of such regimens has beendescribed in the art and may be readily selected.

For example, prime-boost regimens may involve the administration of aDNA (e.g., plasmid) based vector to prime the immune system to second,booster, administration with a traditional antigen, such as a protein ora recombinant virus carrying the sequences encoding such an antigen.See, e.g., WO 00/11140, published Mar. 2, 2000, incorporated byreference. Alternatively, an immunization regimen may involve theadministration of a recombinant SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 vector to boost the immuneresponse to a vector (either viral or DNA-based) carrying an antigen, ora protein. In still another alternative, an immunization regimeninvolves administration of a protein followed by booster with a vectorencoding the antigen.

In one embodiment, a method of priming and boosting an immune responseto a selected antigen by delivering a plasmid DNA vector carrying saidantigen, followed by boosting with a recombinant SAdV-A1321, SAdV-A1325,SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 vector is described.In one embodiment, the prime-boost regimen involves the expression ofmultiproteins from the prime and/or the boost vehicle. See, e.g., R. R.Amara, Science, 292:69-74 (6 Apr. 2001) which describes a multiproteinregimen for expression of protein subunits useful for generating animmune response against HIV and SIV. For example, a DNA prime maydeliver the Gag, Pol, Vif, VPX and Vpr and Env, Tat, and Rev from asingle transcript. Alternatively, the SIV Gag, Pol and HIV-1 Env isdelivered in a recombinant SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 adenovirus construct. Still otherregimens are described in WO 99/16884 and WO 01/54719.

However, the prime-boost regimens are not limited to immunization forHIV or to delivery of these antigens. For example, priming may involvedelivering with a first SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 vector followed by boosting with a second Advector, or with a composition containing the antigen itself in proteinform. In one example, the prime-boost regimen can provide a protectiveimmune response to the virus, bacteria or other organism from which theantigen is derived. In another embodiment, the prime-boost regimenprovides a therapeutic effect that can be measured using conventionassays for detection of the presence of the condition for which therapyis being administered.

The priming composition may be administered at various sites in the bodyin a dose dependent manner, which depends on the antigen to which thedesired immune response is being targeted. The amount or situs ofinjection(s) or to pharmaceutical carrier is not a limitation. Rather,the regimen may involve a priming and/or boosting step, each of whichmay include a single dose or dosage that is administered hourly, daily,weekly or monthly, or yearly. As an example, the mammals may receive oneor two doses containing between about 10 μg to about 50 μg of plasmid incarrier. A desirable amount of a DNA composition ranges between about 1μg to about 10,000 μg of the DNA vector. Dosages may vary from about 1μg to 1000 μg DNA per kg of subject body weight. The amount or site ofdelivery is desirably selected based upon the identity and condition ofthe mammal.

The dosage unit of the vector suitable for delivery of the antigen tothe mammal is described herein. The vector is prepared foradministration by being suspended or dissolved in a pharmaceutically orphysiologically acceptable carrier such as isotonic saline; isotonicsalts solution or other formulations that will be apparent to thoseskilled in such administration. The appropriate carrier will be evidentto those skilled in the art and will depend in large part upon the routeof administration. The compositions described herein may be administeredto a mammal according to the routes described above, in a sustainedrelease formulation using a biodegradable biocompatible polymer, or byon-site delivery using micelles, gels and liposomes. Optionally, thepriming step also includes administering with the priming composition, asuitable amount of an adjuvant, such as are defined herein.

Preferably, a boosting composition is administered about 2 to about 27weeks after administering the priming composition to the mammaliansubject. The administration of the boosting composition is accomplishedusing an effective amount of a boosting composition containing orcapable of delivering the same antigen as administered by the primingDNA vaccine. The boosting composition may be composed of a recombinantviral vector derived from the same viral source (e.g., adenoviralsequences of the invention) or from another source. Alternatively, the“boosting composition” can be a composition containing the same antigenas encoded in the priming DNA vaccine, but in the form of a protein orpeptide, which composition induces an immune response in the host. Inanother embodiment, the boosting composition contains a DNA sequenceencoding the antigen under the control of a regulatory sequencedirecting its expression in a mammalian cell, e.g., vectors such aswell-known bacterial or viral vectors. The primary requirements of theboosting composition are that the antigen of the composition is the sameantigen, or a crossreactive antigen, as that encoded by the primingcomposition.

In another embodiment, the SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 vectors are also well suited foruse in a variety of other immunization and therapeutic regimens. Suchregimens may involve delivery of SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 vectors simultaneously orsequentially with Ad vectors of different serotype capsids, regimens inwhich SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 vectors are delivered simultaneously or sequentially withnon-Ad vectors, regimens in which the SAdV-A1321, SAdV-A1325,SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 vectors are deliveredsimultaneously or sequentially with proteins, peptides, and/or otherbiologically useful therapeutic or immunogenic compounds. Such uses willbe readily apparent to one of skill in the art.

In still another embodiment, the invention provides the use of capsid ofthese viruses (optionally an intact or recombinant viral particle or anempty capsid is used) to induce an immunomodulatory effect response, orto enhance or adjuvant a cytotoxic T cell response to another activeagent by delivering an adenovirus SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 capsid to a subject. TheSAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 capsid can be delivered alone or in a combination regimenwith an active agent to enhance the immune response thereto.Advantageously, the desired effect can be accomplished without infectingthe host with a subgroup E adenovirus. In another aspect, a method ofinducing interferon alpha production in a subject in need thereofcomprising delivering the SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 capsid to a subject is provided.In still another aspect, a method for producing one or more cytokines(e.g., IFN-α)/chemokines in culture is provided. This method involvesincubating a culture containing dendritic cells and the SAdV-A1321,SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 capsiddescribed herein under conditions suitable to producecytokines/chemokines, including, alpha interferon, among others.

The cytokines so produced are useful in a variety of applications. Forexample, in the case of IFNα, the production described herein isparticularly desirable, as it is believed that it will provideadvantages over commercially available recombinantly produced IFNα,which contain only one or two subtypes of IFNα produced in bacteria. Incontrast, the method is anticipated to produce multiple subtypes ofnatural human IFNα, which is expected to result in a broader spectrum ofaction. It is believed that each subtype employs a specific biologicalactivity. Further, it is anticipated that the natural interferonproduced by the method provided herein will be immunologicallyindistinguishable from the patient's naturally produced interferon,thereby reducing the risk of the drug being rejected by the subject'simmune system, usually caused by the formation of neutralizingantibodies against recombinantly produced interferons.

Other cytokines produced by the subgroup E adenoviruses include,interleukin (IL)-6, IL-8, IP-10, macrophage inflammatory protein-1 alpha(MIP-1α), RANTES, and tumor necrosis factor alpha. Methods of purifyingthese cytokines/chemokines from culture and therapeutic or adjuvant usesof these cytokines/chemokines have been described in the literature.Further, commercially available columns or kits may used forpurification of the cytokines/chemokines prepared according to theinvention. The cytokines/chemokines produced using the invention may beformulated for use in a variety of indications.

For example, cytokines described herein include, interferon alpha(IFNα), tumor necrosis factor alpha (TNFα), IP-10 (Interferon gammaInducible Protein), interleukin-6 (IL-6), and IL-8. IFNα, has beendescribed as being useful in treatment of influenza, hepatitis(including, e.g., hepatitis B and C), and a variety of neoplasms, e.g.,kidney (renal cell carcinoma), melanoma, malignant tumor, multiplemyeloma, carcinoid tumor, lymphoma and leukemia (e.g., chronicmyelogenous leukemia and hairy cell leukemia). A mixture of IFNαsubtypes produced as described herein can be purified using knowntechniques. See, e.g., WO 2006/085092, which describes the use ofmonoclonal antibodies and column purification. Other techniques havebeen described in the literature. IFNα produced as described herein canbe purified using known methods. See, e.g., U.S. Pat. No. 4,680,260,U.S. Pat. No. 4,732,683, and G. Allen, Biochem J., 207:397-408 (1982).TNFα has been described as being useful in treatment in autoimmunedisorders including, e.g., psoriasis and rheumatoid arthritis. IP-10,Interferon gamma Inducible Protein, can be used as a potent inhibitor ofangiogenesis and to have a potent thymus-dependent anti-tumor effect.

A method for producing IFNα by incubating a culture containing dendriticcells and a SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316,or SAdV-A1322 capsid under conditions suitable to produce cytokines isprovided. In one embodiment, blood is drawn from healthy donors(preferably human) and peripheral blood leukocytes (PBL) or peripheralblood mononuclear cells (PBMC) are prepared using known techniques. Inone embodiment, PBL are used as the cytokine-producing cells accordingto the method of the invention. In another embodiment, PBMC are used asthe cytokine-producing cells. In another embodiment, plasmacytoiddendritic cells are isolated from the PBL or PBMC using knowntechniques, e.g., using the commercially available kit “humanplasmacytoid dendritic cell isolation kit” by Miltenyi Biotec GmbH(Germany). The selected cells are cultured in suspension with anappropriate media and the adenovirus subgroup E capsid protein.Appropriate media can be readily determined by one of skill in the art.However, in one embodiment, the media is a RPMI-1640 medium.Alternatively, other media may be readily selected. The cells may becultured in a suitable vessel, e.g., a microtiter well, a flask, or alarger vessel. In one embodiment, the concentration of the cells isabout 1 million cells/mL culture media. However, other suitable cellconcentrations may be readily determined by one of skill in the art. Theinvention does not require the use of interferons as primers. However,if desired, the media may include a suitable cytokine, IL-3, in order tostimulate cell growth. One suitable concentration is about 20 ng/mL.However, other concentrations may be used. In one embodiment, theSAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, orSAdV-A1322 capsid protein is introduced into the culture containing thecells. The adenovirus capsid protein can be delivered to the culture inany of the forms described herein (e.g., a viral particle, including anempty capsid particle, a viral vector having an SAdV-A1321, SAdV-A1325,SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 capsid, and the like).Typically the capsid protein will be suspended in a suitable carrier,e.g., culture media, saline, or the like. Suitably, the SAdV-A1321,SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 capsid isadded to the culture in an amount of about 100 to 100,000 adenovirussubgroup E particles per cell. The mixture is then incubated, e.g., inthe range of about 28° C. to about 40° C., in the range from about 35°C. to about 37° C., or about 37° C. Typically, approximately 12 to 96hours, or about 48 hours later, cells are spun down and the supernatantis collected. Suitably, this is performed under conditions which avoidcell lysis, thereby reducing or eliminating the presence of cellulardebris in the supernatant. Centrifugation permits separation of thecytokines from the cells, thereby providing a crudely isolated cytokine.Sizing columns, and other known columns and methods are available forfurther purification of cytokines from adenoviruses and adenoviralcapsids, and the like. These cytokines, so purified, are available forformulation and use in a variety of applications.

In one embodiment, an empty SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 particle (i.e., an adenoviralcapsid having no DNA packaged therein which expresses any adenoviral ortransgene product) may be delivered to the cells. In another embodiment,a non-infectious wild-type SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 particle or a recombinantadenoviral vector packaged in an SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 capsid (particle) may be used.Suitable techniques for inactivating such viral particles are known inthe art and may include without limitation, e.g., UV irradiation (whicheffectively cross-links genomic DNA preventing expression).

The following examples describe the cloning of SAdV-A1321, SAdV-A1325,SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 and the constructionof exemplary recombinant SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309,SAdV-A1316, or SAdV-A1322 vectors. These examples are illustrative only,and do not limit the scope of the present invention.

Example 1 Isolation of Simian Adenoviruses

Stool samples were obtained from the chimpanzee colony at the Universityof Louisiana New Iberia Research Center, 4401 W. Admiral Doyle Drive,New Iberia, La., USA. Filtered supernatants from the stool suspensionswere inoculated into cultures of the human cell line A549. After about 1to 2 weeks in culture, visual cytopathic effect (CPE) was obvious incell cultures with several of the inocula. The viruses that wereisolated by this technique were amplified to a large-scale preparationusing A549 cells using the standard adenovirus purification method ofcesium chloride gradient banding. DNA from the purified adenoviruses wasisolated and completely sequenced by Qiagen Genomics services, Hilden,Germany. Analysis of the complete genomic sequence showed that theisolated virus had a novel sequence that had not been previouslyreported.

Based on the phylogenetic analysis of the viral DNA sequences, theadenoviruses designated simian adenovirus A1321 (SAdV-A1321),SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316, and SAdV-A1322, weredetermined to be in the same subgroup as subgroup (species) E. Averageyields for viral amplification were as follows: A1321 (1.44×10¹³),SAdV-A1325 (2.73×10¹³), SAdV-A1295 (1.2×10¹³), SAdV-A1309 (2.57×10¹³),SAdV-A1316 (5.58×10¹³), and SAdV-A1322 (4.71×10¹³).

Example 2 Vector Construction

An E1 deleted vector using SAdV-A1321, SAdV-A1325, SAdV-A1295,SAdV-A1309, SAdV-A1316, or SAdV-A1322 (subgroup E) may be preparedgenerally as described.

A linker containing SmaI, ClaI, XbaI, SpeI, EcoRV sites flanked by SwaIis cloned into pBR322 cut with EcoRI and NdeI. Viral DNA is digestedwith XbaI and the 6 kb fragments (left and right ends) are gel purifiedand ligated into pSR5 digested with SmaI and XbaI. 12 minipreps arediagnosed with SmaI and assessed for expected fragment sizes. Miniprepsare sequenced to check the integrity of the viral DNA end. The sequenceobtained is used to correct the left end Qiagen sequence and deduce thecorrect right ITR sequence as well.

The plasmid is digested with SnaBI+NdeI and the NdeI site is filled inwith Klenow. The EcoRV fragment from pBleuSK I-PI is ligated in.Alternatively the plasmid is digested by SnaBI and NdeI and a doublestranded oligonucleotide containing recognition sites for CeuI andPI-SceI is ligated in place of deleted E1 coding regions. Minipreps arediagnosed using PstI. The resulting plasmid is digested with XbaI+EcoRV.The right end (XbaI digest) fragment from the SAdV-A1321, SAdV-A1325,SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 is ligated in.Minipreps are diagnosed using ApaLI. The resulting plasmid is thendigested with XbaI+EcoRV. The fragment from the SAdV-A1321, SAdV-A1325,SAdV-A1295, SAdV-A1309, SAdV-A1316, or SAdV-A1322 DNA is ligated in andminipreps are diagnosed using MfeI. 293 cells are then transfected usingcalcium phosphate or lipofectamine methods according to manufacturer'sprotocol.

Example 3 Assessment of Cross-Neutralizing Antibodies

A. Wild-type SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316,and SAdV-A1322 are assessed for cross-neutralizing activity as comparedto human Adenovirus 5 (subspecies C) and chimpanzee adenovirus 7(SAdV-24), and human pooled IgG using an infection inhibitionneutralizing antibody assay monitored by direct immunofluorescence. Thehuman pooled IgG [Hu Pooled IgG] is purchased commercially and isapproved for administration in immunocompromised patients, as itcontains antibodies against a number of antigens to which the generalhuman population is exposed. The presence or absence of neutralizingantibodies to the simian adenoviruses for the human pooled IgG is areflection of the prevalence of antibodies to these adenoviruses in thegeneral population.

The assay is performed as follows. Serum samples obtained from rabbitspreviously injected with HAdV-5 or SAdV-24 are heat inactivated at 56°C. for 35 min. Wild type adenovirus (10⁸ particles/well) are diluted inserum-free Dulbecco's modified Eagle's medium (DMEM) and incubated with2-fold serial dilutions of heat-inactivated serum samples in DMEM for 1h at 37° C. Subsequently, the serum-adenovirus mixture is added toslides in wells with 105 monolayer A549 cells. After 1 hr, the cells ineach well are supplemented with 100 μl of 20% fetal bovine serum(FBS)-DMEM and cultured for 22 h at 37° C. in 5% CO₂. Next, cells arerinsed twice with PBS and stained with DAPI and a goat, FITC labeled,broadly cross reactive antibody (Virostat) raised against HAdV-5following fixation in paraformaldehyde (4%, 30 min) and permeabilizationin 0.2% Triton (4° C., 20 min) The level of infection is determined bycounting the number of FITC positive cells under microscopy. The NABtiter is reported as the highest serum dilution that inhibits adenovirusinfection by 50% or more, compared with the naive serum control. Where atiter value of < 1/20 is shown, the neutralizing antibody concentrationis under the limit of detection, i.e., 1/20.

B. Wild-type SAdV-A1321, SAdV-A1325, SAdV-A1295, SAdV-A1309, SAdV-A1316,and SAdV-A1322 were assessed for cross-neutralizing activity as comparedto human Adenovirus 5 (HAdV-5; subspecies C). The results are shown inTable 3 below. Less than approximately 15% of the population of humansamples (n=20) had a neutralizing antibody titer (NAb titer) greaterthan 200 for the identified adenoviruses, relative to approximately 40%for HAdV-5.

TABLE 3 Human samples Wild type IVIG Nab titer (n = 20) Nab titeradenovirus (10 mlg/ml) Median Mean A1295 20 20 40 A1309 80 60 113 A131640 80 91 A1321 20 40 40 A1322 40 40 69 A1325 80 80 88 HAdV-5 640 6401589

Example 4 Vector Construction

A. SAdV-A1321

An E1 deleted SAdV-A1321 vector is prepared by digesting the pSR5plasmid (SEQ ID NO: 322) with SmaI+XbaI, and the wild-type SAdV-A1321sequence (SEQ ID NO: 1) with Xba I to produce an 6020 bp fragment forincorporation into the plasmid. The resulting plasmid (pS215-1321) isdigested with BsiWI+NdeI or with SnaBI+NdeI, and ICeuPISceI meganucleasecassette(s) (SEQ ID NO: 326) cloned therein via SnaIB (BsiWI)+NdeIsites. The resulting plasmid (pS216-1321) is then digested withEcoRV+XbaI, and the ˜30526 bp fragment of wild-type SAdV-A1321 sequence(SEQ ID NO: 1) digested with XbaI is cloned therein resulting in thepS217-A1321 plasmid.

A suitable transgene expression cassette is then introduced into thepS217-A1321 plasmid. The transgene may be, e.g., a reporter such aseGFP, an influenza A nucleoprotein, or HIV-gag (e.g., frompSh-HIV-short-gag (SEQ ID NO: 319)) via the I-CeuI and PI-SceI sites ofthe meganuclease cassette. Additional transgenes described herein andknown in the art may be used consistent with this example and the skillin the art and are contemplated hereby.

A proposed E1 deleted SAdV-A1321 vector containing an HIV-gag transgeneis identified in SEQ ID NO: 168.

B. SAdV-A1325

An E1 deleted SAdV-A1325 vector is prepared by digesting the pSR5plasmid (SEQ ID NO: 322) with SmaI+XbaI, and the wild-type SAdV-A1325sequence (SEQ ID NO: 28) with Xba I to produce an 5728 bp fragment forincorporation into the plasmid. The resulting plasmid (pS226-1325) isdigested with BsiWI+NdeI or with SnaBI+NdeI, and ICeuPISceI meganucleasecassette(s) (SEQ ID NO: 326) cloned therein via SnaIB (BsiWI)+NdeIsites. The resulting plasmid (pS227-1325) is then digested withEcoRV+XbaI, and the ˜30507 bp fragment of wild-type SAdV-A1325 sequence(SEQ ID NO: 28) digested with XbaI is cloned therein resulting in thepS228-A1325 plasmid.

A suitable transgene expression cassette is then introduced into thepS228-A1325 plasmid. The transgene may be, e.g., a reporter such aseGFP, an influenza A nucleoprotein, or HIV-gag (e.g., frompSh-HIV-short-gag (SEQ ID NO: 319)) via the I-CeuI and PI-SceI sites ofthe meganuclease cassette. Additional transgenes described herein andknown in the art may be used consistent with this example and the skillin the art and are contemplated hereby.

A proposed E1 deleted SAdV-A1325 vector containing an HIV-gag transgeneis identified in SEQ ID NO: 193.

C. SAdV-A1295

An E1 deleted SAdV-A1295 vector is prepared by digesting the pSR5plasmid (SEQ ID NO: 322) with SmaI+XbaI, and the wild-type SAdV-A1295sequence (SEQ ID NO: 57) with Xba I to produce an ˜6017 bp fragment forincorporation into the plasmid. The resulting plasmid (pS200-1295) isdigested with BsiWI+NdeI and the ends are filled in with Klenow andtreated with CIP. The pBleuSK I-PI plasmid (SEQ ID NO: 324) is digestedwith EcoRV and the EcoRV fragment from pBleuSK I-PI (harboring sites forI-CeuI and PI-SceI) was ligated in. The resulting plasmid (pS201-1295)is then digested with EcoRV+XbaI, and the ˜6521 bp fragment of wild-typeSAdV-A1295 sequence (SEQ ID NO: 57) digested with XbaI is cloned thereinresulting in the pS202_A1295 plasmid. The pS202-A1295 plasmid is thendigested with XbaI and the 24105 bp fragment of wild-type SAdV-A1295sequence (SEQ ID NO: 57) digested with XbaI is cloned therein resultingin the pS203-A1295 plasmid.

A suitable transgene expression cassette is then introduced into thepS203-A1295 plasmid. The transgene may be, e.g., a reporter such aseGFP, an influenza A nucleoprotein, or HIV-gag (e.g., frompSh-HIV-short-gag (SEQ ID NO: 319)) via the I-CeuI and PI-SceI sites ofthe pBleuSK I-PI plasmid fragment. Additional transgenes describedherein and known in the art may be used consistent with this example andthe skill in the art and are contemplated hereby.

A proposed E1 deleted SAdV-A1295 vector containing an HIV-gag transgeneis identified in SEQ ID NO: 220.

D. SAdV-A1309

An E1 deleted SAdV-A1309 vector is prepared by digesting the pSR5plasmid (SEQ ID NO: 322) with SmaI+XbaI, and the wild-type SAdV-A1309sequence (SEQ ID NO: 86) with Xba I to produce an 6037 bp fragment forincorporation into the plasmid. The resulting plasmid (pS205-1309) isdigested with NsiWI+NedI or with SnaBI+NdeI and ICeuPISceI meganucleasecassette(s) (SEQ ID NO: 326) cloned therein via SnaIB (BsiWI)+NdeIsites. The resulting plasmid (pS206-1309) is then digested withFse+EcoRV, and the ˜1924 bp Fse-end fragment of wild-type SAdV-A1309sequence (SEQ ID NO: 86) is cloned therein resulting in the pS207-A1309plasmid. pS207-A1309 is digested with Fse and the ˜17,731 bp Fse-Fsefragment of wild-type SAdV-A1309 sequence (SEQ ID NO: 86) is clonedtherein resulting in the pS208-A1309 plasmid. pS208 is digested with Speand the ˜24,718 bp Spe-Spe fragment of wild-type SAdV-A1309 sequence(SEQ ID NO: 86) is cloned therein resulting in the pS209-A1309 plasmid.

A suitable transgene expression cassette is then introduced into thepS209-A1309. The transgene may be, e.g., a reporter such as eGFP, aninfluenza A nucleoprotein, or HIV-gag (e.g., from pSh-HIV-short-gag (SEQID NO: 319)) via the I-CeuI and PI-SceI sites of the meganucleasecassette. Additional transgenes described herein and known in the artmay be used consistent with this example and the skill in the art andare contemplated hereby.

A proposed E1 deleted SAdV-A1309 vector containing an HIV-gag transgeneis identified in SEQ ID NO: 246.

E. SAdV-A1316

An E1 deleted SAdV-A1316 vector is prepared by digesting the pSR7plasmid (SEQ ID NO: 323) with SnaBI+NheI, and the wild-type SAdV-A1316sequence (SEQ ID NO: 114) with NheI to produce an ˜3032 bp fragment forincorporation into the plasmid. The resulting plasmid (pS210-A1316) isdigested with NsiWI+NedI or with SnaBI+NdeI and ICeuPISceI meganucleasecassette(s) (SEQ ID NO: 326) cloned therein via SnaIB (BsiWI)+NdeIsites. The resulting plasmid (pS211-A1316) was digested with NheI+EcoRVand the ˜771 bp Nhe digested fragment of the wild-type SAdV-A1316sequence (SEQ ID NO: 114) cloned in. Similarly, the resulting plasmidpS212-A1316 is digested with NheI and the 32845 bp Nhe digested fragmentof the wild-type SAdV-A1316 sequence (SEQ ID NO: 114) cloned in(resulting in pS213-A1316).

A suitable transgene expression cassette is then introduced into thepS213-A1306 plasmid. The transgene may be, e.g., a reporter such aseGFP, an influenza A nucleoprotein, or HIV-gag (e.g., frompSh-HIV-short-gag (SEQ ID NO: 319)) via the I-CeuI and PI-SceI sites ofthe meganuclease cassette. Additional transgenes described herein andknown in the art may be used consistent with this example and the skillin the art and are contemplated hereby.

A proposed E1 deleted SAdV-A1316 vector containing an HIV-gag transgeneis identified in SEQ ID NO: 272.

F. SAdV-A1322

A proposed E1 deleted SAdV-A1322 vector containing an HIV-gag transgeneis identified in SEQ ID NO: 295, and may be prepared according asindicated above by one of ordinary skill in the art. [An L1 IIIa regionis contained at nt 12273-13776, which is not codable within the SequenceListing.]

Example 5 T-Cell Induction

The protocols contained in Roy, et al. [“Partial protection against H5N1influenza in mice with a single dose of a chimpanzee adenovirus vectorexpressing nucleoprotein”, Vaccine 25:6845-6851 (Aug. 6, 2007)], whichis herein incorporated by reference, may be utilized to assess T cellinduction by the resulting recombinant adenovirus virus.

Example 6 Cytokine Induction

Characterization of cytokine responses to adenoviral vectors describedhere is performed according to the methods of Lin, et al., J Virol. 2007November; 81(21): 11840-11849 (Vaccines Based on Novel Adeno-AssociatedVirus Vectors Elicit Aberrant CD8⁺ T-Cell Responses in Mice), and Lin,et al., Hum. Gene Ther. 2008 July; 19(7): 663-669 (Impact of PreexistingVector Immunity on the Efficacy of Adeno-Associated Virus-Based HIV-1Gag Vaccines), including Enzyme-linked immunosorbent assay, Interferon-γenzyme-linked immunospot assay, and Intracellular cytokine staining(ICCS).

Characterization is expected to reflect an advantageous cytokine profilefollowing vector administration.

All documents recited above, the Sequence Listing, and the entirety ofInternational Patent Application No. PCT/US2011/061632 and U.S.Provisional Patent Application Nos. 61/416,467, 61/416,481, 61/416,491,61/416,499, 61/416,509, and 61/416,515 (all filed Nov. 23, 2010) areincorporated herein by reference. Numerous modifications and variationsare included in the scope of the above-identified specification and areexpected to be obvious to one of skill in the art. Such modificationsand alterations to the compositions and processes, such as selections ofdifferent minigenes or selection or dosage of the vectors or immunemodulators are believed to be within the scope of the claims appendedhereto.

1. An adenovirus having a capsid comprising a hexon protein, a pentonprotein, and a fiber protein, wherein said hexon protein is the hexonprotein of SAdV-A1295 with the amino acids 1 to 940 of SEQ ID NO: 67;said capsid encapsidating a heterologous nucleic acid comprising a geneoperably linked to expression control sequences which directtranscription, translation, and/or expression thereof in a host cell. 2.The adenovirus according to claim 1, further comprising a 5′ and a 3′adenovirus cis-element necessary for replication and encapsidation. 3.The adenovirus according to claim 1, wherein said adenovirus lacks allor a part of the E1 gene.
 4. The adenovirus according to claim 3,wherein said adenovirus is replication-defective.
 5. The adenovirusaccording to claim 1, wherein said penton protein is the penton proteinof SAdV-A1295 with the amino acids 1 to 534 of SEQ ID NO:
 62. 6. Theadenovirus according to claim 1, wherein said fiber protein is the fiberprotein of SAdV-A1295 with the amino acids 1 to 442 of SEQ ID NO:
 78. 7.The adenovirus according to claim 1, wherein said capsid is a hybridcapsid.
 8. The adenovirus according to claim 7, wherein said hybridcapsid comprises at least one capsid protein from an adenovirus selectedfrom SAdV-A1321, SAdV-A1325, SAdV-A1316, and SAdV-1322.
 9. A compositioncomprising the adenovirus according to claim 1 in a pharmaceuticallyacceptable carrier.
 10. A method for targeting a cell having anadenoviral receptor comprising delivering to a subject a virus accordingto claim
 1. 11. A vector comprising at least one simian adenovirusnucleic acid sequence selected from the group consisting of: (a) theopen reading frame for the penton of SAdV-A1295, nucleotides 13857 to15458 of SEQ ID NO: 57; (b) the open reading frame for the hexon ofSAdV-A1295, nucleotides 18290 to 21109 of SEQ ID NO: 57; and (c) theopen reading frame for the fiber protein of SAdV-A1295, nucleotides32227 to 33552 of SEQ ID NO: 57; said vector further comprising aheterologous gene operably linked to expression control sequences.
 12. Acomposition comprising the adenovirus according to claim 1, saidadenovirus further comprising at least one simian adenovirus proteinselected from the group consisting of: E1a, SEQ ID NO: 85; E1b, smallT/19K, SEQ ID NO: 58; E1b, large T/55K, SEQ ID NO: 80; IX, SEQ ID NO:59; 52/55D, SEQ ID NO: 60; Ma, SEQ ID NO: 61; Penton, SEQ ID NO: 62;VII, SEQ ID NO: 63; V, SEQ ID NO: 64; pX, SEQ ID NO: 65; VI, SEQ ID NO:66; Endoprotease, SEQ ID NO: 68; 100 kD, SEQ ID NO: 69; 22 kD, SEQ IDNO: 81; VIII, SEQ ID NO: 70; E3/12.5 K, SEQ ID NO: 71; CR1-alpha SEQ IDNO: 82; gp19K, SEQ ID NO: 72; CR1-beta, SEQ ID NO: 73; CR1-gamma, SEQ IDNO: 74; CR1-delta, SEQ ID NO: 75; RID-alpha, SEQ ID NO: 76 RID-beta, SEQID NO: 77; E3/14.7K, SEQ ID NO: 83; and Fiber, SEQ ID NO: 78.